CN107760624A - 一种产乙醛脱氢酶的植物乳杆菌及其应用 - Google Patents
一种产乙醛脱氢酶的植物乳杆菌及其应用 Download PDFInfo
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Abstract
本发明涉及微生物技术领域，尤其涉及一种产乙醛脱氢酶植物乳杆菌及其应用。该植物乳杆菌命名为植物乳杆菌FCJX 102(lactobacillus plantarum FCJX 102)，已于2017年6月16日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO:M2017340。植物乳杆菌FCJX 102(lactobacillus plantarum FCJX 102)优点在于，乙醇诱导下可高产乙醛脱氢酶，且在解酒护肝药品与食品方面的具有广阔的应用前景。
Description
技术领域
本发明涉及微生物技术领域，尤其涉及一种产乙醛脱氢酶植物乳杆菌及其应用。
背景技术
乳酸菌是一类革兰氏阳性、利用可发酵糖产生大量乳酸的细菌的通称。乳酸菌广泛地存在于自然界、动植物体、乳制品和发酵食品中,且大多数为非致病菌,属于有益微生物,是人体正常菌群。
植物乳酸菌是通过蔬菜发酵后分离出来的乳酸菌，植物乳杆菌。它可以抑制肠道中腐败菌的生长，促进肠道中双歧杆菌的增殖，可改善肠道环境而有利于健康。而植物乳酸菌在肠道中可以制造人体所需的维生素VK、VB6、VB12、RIBOFLAVIN、叶酸等，同时可以抑制破坏维生素B1酵素，使肠道内维生素B族保持稳定。国内现有的文献中记载的植物乳酸菌有多种，功能多种多样，具有多领域的应用，但是目前在国内并没有报道植物乳酸菌具有产乙醛脱氢酶功能的文献。
发明内容
本发明提供一种能产乙醛脱氢酶的植物乳杆菌及其应用，该植物乳杆菌在解酒药品以及食品方面的应用有极大的市场前景。
本发明为在解酒药品以及食品方面的研究提供参考。
技术方案：
本发明提供的一种植物乳杆菌FCJX 102(lactobacillus plantarum FCJX 102)，已于2017年6月16日保藏于中国典型培养物保藏中心，保藏单位地址：中国武汉武汉大学，保藏号为CCTCC NO:M2017340。
植物乳杆菌FCJX 102自发酵旺盛期的泡菜发酵液中筛选分离得到，通过一系列实验得到植物乳杆菌FCJX 102的形态特征，生理特性以及rDNA-ITS2序列：
形态特征：菌体呈杆状，菌体大小一般为0.3-3um，菌株在 MRS平板上划线分离，37℃培养，菌株生长良好，其菌落形态如图1所示。菌落大小0.2-2um，菌落圆形，边缘整齐，正面微凸起，颜色乳白色，不透明，表面湿润光滑。植物乳杆菌的最低生长温度为15℃，最高生长温度为45℃，在30-40℃生长温度最佳，菌株的延迟期相对较短，2h进入对数生长期，12h达到稳定期。
生理特性：从生理生化结果可知，植物乳杆菌FCJX 102为革兰氏染色阳性、细胞形状杆菌，触酶试验阴性，凝乳实验表明具有产酸能力，初步判断为乳杆菌。
rDNA-ITS2序列：测序所得的植物乳杆菌FCJX 102的 rDNA-ITS2序列与同源性NCBI BLAST比对分析，植物乳杆菌 FCJX 102的rDNA-ITS2序列与GenBank中乳杆菌
(ID:KP174109.1)的rDNA-ITS2序列相似度达到99％。
由植物乳杆菌FCJX 102的形态特征，生理特性以及 rDNA-ITS2序列得到植物乳杆菌FCJX 102属于乳杆菌目、乳杆菌科、乳杆菌属、植物乳杆菌种。
与现有技术相比，本发明的优点在于：
(1)目前，国内外均未有关于产乙醛脱氢酶的植物乳杆菌的报道。
(2)本发明所述植物乳杆菌FCJX 102可产乙醛脱氢酶，经乙醇诱导后高产乙醛脱氢酶，其在解酒药品以及食品方面的应用有极大的市场前景。
附图说明
图1植物乳杆菌FCJX 102的划线培养图
具体实施方式
以下通过实施例对本发明进行进一步的说明。
实施例1 MRS培养基的制备
称取蛋白胨10.0g，牛肉粉5.0g，酵母浸粉4.0g，葡萄糖 20.0g，乙酸钠5.0g，柠檬酸氢二铵2.0g，磷酸氢二钾2.0g，七水硫酸镁0.2g，硫酸锰0.05g于锥形瓶中，再加入吐温-80 1.0mL、水1000mL混匀，将pH调至6.2～6.4，115℃灭菌30min 即可。
实施例2从泡菜筛选植物乳杆菌
(1)取发酵旺盛期的泡菜发酵液，将4只1.5mL离心管分别编号为1、2、3、4，用移液枪取0.1mL微生态制剂样品于1 号离心管中,TGL-16B高速台式离心机中10000rpm离心1min, 弃上清。加入200μL生理盐水清洗，将离心管放入WH-866旋涡混合器中旋涡振荡混匀。然后，再次10000rpm离心1min,弃上清。
(2)用移液枪取1000μL生理盐水于1号离心管中，上下颠倒数次混匀，所得的菌液即稀释为10-1。其它3支离心管各加 900μL生理盐水。从1号离心管中取100μL菌液于2号管中，上下颠倒数次混匀，所得的菌即稀释为10-2。同理，以此类推，3 号管稀释为10-3，4号管稀释为10-4。
(3)从1、2、3、4号管中各取稀释液0.2mL，用涂布法接种乳酸菌分离培养基中。37℃于HWS-250型恒温恒湿培养箱培养48h，形成菌落后观察其形态，挑取周围变色圈的菌落，进行多次平板划线分离得到纯化的单菌落。将筛选得到的4株单菌落进行斜面保藏，得到的4株单菌名称为乳杆菌FCJX 101、FCJX 102、FCJX 103、FCJX 104。
实施例3植物乳杆菌的生理生化的鉴定
将4株为乳杆菌FCJX 101、FCJX 102、FCJX 103、FCJX 104 画线于MRS固体培养基中37℃恒温培养48h后分别做以下实验：
(1)革兰氏染色
a.涂片取洁净玻片一块，作好标记，用胶头滴管滴加一生理盐水置玻片上；用接种针挑取少许单菌落，和玻片上的生理盐水混匀涂片。
b.干燥固定：将涂好的玻片在酒精灯火焰上方来回烘烤约半分钟，使玻片上的细菌悬液干燥并固定。
c.染色：滴加结晶紫染液一滴，染色1min，用水冲洗，接着用碘液作用1min，用水冲洗；然后用95％乙醇脱色，直至流出的乙醇无紫色时，立即水洗。最后用蕃红复染约1min，用水冲洗，于室温下晾干。
d.镜下观察：100倍油镜下观察革兰氏染色结果及显微镜下细菌形态。
(2)凝乳实验
用灭菌接种针挑取MRS固体培养基中的菌落接种到脱脂乳培养基中，37℃在HWS-250型恒温恒湿培养箱培养48h，观察凝乳情况及凝乳特性。
(3)触酶试验
用接种环挑取MRS固体培养基上菌落，置于洁净玻片上，滴加3％过氧化氢溶液数滴，观察结果。于半分钟内有大量气泡产生者为阳性，不产气泡者为阴性。
表1乳酸菌的生理生化鉴定结果
从生理生化结果可知，乳杆菌FCJX 101、FCJX 102、FCJX 103、FCJX 104均为革兰氏染色阳性、细胞形状杆菌，触酶试验阴性，凝乳实验表明具有产酸能力，初步判断为乳杆菌。
实施例4乳杆菌产乙醛脱氢酶实验
(1)菌种活化
将保存的乳杆菌FCJX 101、FCJX 102、FCJX 103、FCJX 104 培养于MRS培养基中传代2-3次。
(2)发酵产酶
将培养好的各乳酸菌种子液按5％的接种量接种于MRS培养基中，37℃恒温培养16h。(3)粗酶液制备
株菌的发酵液经4500rpm、4℃离心15min后，沉淀用灭菌生理盐水洗涤2次，收集菌泥，再将菌泥和磷酸盐缓冲液(pH 8.0、 20mmol/L)以1:5的比例制成菌悬液，冰浴中以200W功率超声破碎15min，再于6000rpm、4℃离心15min，收集上清液，沉淀再同样超声处理1次，合并2次上清液即得粗酶液。用下述方法测得乙醛脱氢酶活性。选出具有产酶能力的菌种进行后续实验。
(4)乙醛脱氢酶酶活测定方法
比色皿中加入2.52mL Tris-HCl缓冲液(0.05mol/L，pH＝8.0)、 0.1mL EDTA溶液(0.05mol/L)、0.1mL KCl溶液(3mol/L)、 0.1mL NAD+溶液(0.02mol/L)、0.05mL乙醛溶液(0.1mol/L)、 0.03mLβ-巯基乙醇溶液(0.05mol/L)，混匀后，加入0.1mL 样液，迅速混匀后，于340nm波长下测吸光值，计算5min内吸光值的变化ΔA340。
酶活力单位定义：25℃下，每分钟催化还原lμmol NAD+ 的所需要的酶量为1个酶活力单位。
表2不同植物乳杆菌乙醛脱氢酶酶活结果
分析表2发现，乳杆菌FCJX 101、FCJX 102、FCJX 103、 FCJX 104在乳杆菌的乙醛脱氢酶酶活测定结果中，乳杆菌FCJX 102的在四次酶活结果测定中，其酶活量均为最大，故乳杆菌 FCJX 102为4株筛选出来的乳杆菌中产乙醛脱氢酶最大的乳杆菌。
实施例5乙醇诱导植物乳杆菌FCJX 102产乙醛脱氢酶
将植物乳杆菌FCJX 102按5％接种量接种于MRS培养基中，并添加0％-8％乙醇作为诱导剂，按5％接种量接种于MRS 培养基中37℃培养16h后，按上述方法测定乙醛脱氢酶酶活。
表3植物乳杆菌FCJX 102在不同乙醇浓度下产乙醛脱氢酶能力
分析表3发现，植物乳杆菌FCJX 102经0％-8％乙醇诱导后培养，其产乙醛脱氢酶的酶量在乙醇浓度为0％-4％的区间中呈现先增后减的趋势，在乙醇浓度为0.5％时产酶量达到最大。
序列表
<110> 南昌大学
<120> 一种产乙醛脱氢酶的植物乳杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1159
<212> DNA
<213> 植物乳杆菌 FCJX 102(lactobacillus plantarum FCJX 102)
<400> 1
ttgtcacctt acgcggctgg ttcctaaaag gttaccccac cgactttggg tgttacaaac 60
tctcatggtg tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cggcatgctg 120
atccgcgatt actagcgatt ccgacttcat gtaggcgagt tgcagcctac aatccgaact 180
gagaatggct ttaagagatt agcttactct cgcgagttcg caactcgttg taccatccat 240
tgtagcacgt gtgtagccca ggtcataagg ggcatgatga tttgacgtca tccccacctt 300
cctccggttt gtcaccggca gtctcaccag agtgcccaac ttaatgctgg caactgataa 360
taagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacaac 420
catgcaccac ctgtatccat gtccccgaag ggaacgtcta atctcttaga tttgcatagt 480
atgtcaagac ctggtaaggt tcttcgcgta gcttcgaatt aaaccacatg ctccaccgct 540
tgtgcgggcc cccgtcaatt cctttgagtt tcagccttgc ggccgtactc cccaggcgga 600
atgcttaatg cgttagctgc agcactgaag ggcggaaacc ctccaacact tagcattcat 660
cgtttacggt atggactacc agggtatcta atcctgtttg ctacccatac tttcgagcct 720
cagcgtcagt tacagaccag acagccgcct tcgccactgg tgttcttcca tatatctacg 780
catttcaccg ctacacatgg agttccactg tcctcttctg cactcaagtt tcccagtttc 840
cgatgcactt cttcggttga gccgaaaggc tttcacatca gacttaaaaa accgcctgcg 900
ctcgctttac gcccaataaa tccggacaac gcttgccacc tacgtattac cgcggctgct 960
ggcacgtagt tagccgtggc tttctggtta aataccgtca atacctgaac agttactctc 1020
agatatgttc ttctttaaca acagagtttt acgagccgaa acctctcact cacgcggcgt 1080
gctccatcag acttcgttca tgtgagatcc ctactgctgc ctccgtagag ttgggccgtg 1140
tcctcagtcc caatggtgg 1159
Claims (2)
1.一种产乙醛脱氢酶的植物乳杆菌，其命名为植物乳杆菌FCJX102(lactobacillusplantarum FCJX 102)，已于2017年6月16日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO:M2017340。
2.如权利要求1所述的一种产乙醛脱氢酶的植物乳杆菌在制备醒酒药品及食品方面的应用。
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| CN115322932A (zh) * | 2022-08-24 | 2022-11-11 | 天津科技大学 | 一株具有解酒醒酒能力的植物乳植杆菌和应用 |
| CN115322932B (zh) * | 2022-08-24 | 2023-11-28 | 天津科技大学 | 一株具有解酒醒酒能力的植物乳植杆菌和应用 |
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