CN112143667B - 一种高表达乙醛脱氢酶的耐酸植物乳杆菌及其应用 - Google Patents
一种高表达乙醛脱氢酶的耐酸植物乳杆菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种高表达乙醛脱氢酶的耐酸植物乳杆菌及其应用。一种植物乳杆菌(Lactobacillus plantarum)YLA1,所述植物乳杆菌保藏于中国典型培养物保藏中心,保藏日期为2020年7月6日,保藏号为CCTCC NO:M2020289。上述植物乳杆菌既具有高效乙醛脱氢酶活性,又具备乳酸菌耐酸的特性,可有效防止胃酸对其的破坏,能够发挥较长时间的乙醛脱氢酶活性,缓解乙醛刺激导致的脸红不退、心率加速、胃部不适及头疼的酒后症状,可用于增加人体对酒精的耐受力,增进酒精在人体中的代谢速率,具有较好的实际应用价值,可应用于食品,药品等领域解酒产品的开发。
Description
技术领域
本发明属于益生菌健康食品技术领域,具体涉及一种高表达乙醛脱氢酶的耐酸植物乳杆菌及其应用。
背景技术
植物乳杆菌是一种常见的发酵乳酸菌,属于革兰氏阳性细菌,其大量存在于环境中,多见于植物体表。大小为0.9-1.2μm×3.0-8.0μm,不产芽孢,可应用于酸奶、奶酪、发酵调味品、泡菜和香肠等食品生产。其最适生长温度32-38℃,最适pH 6.5。植物乳杆菌可利用葡萄糖和乳糖来产酸,同时可以产生细菌素和双乙酞等天然抑菌物质,从而抑制病原菌的生长调节肠胃功能。研究表明,植物乳杆菌可以促进机体生长,促进蛋白质等营养物质转化,提高钙、磷等矿物质的利用率。此外植物乳杆菌基因组中具备编码乙醛脱氢酶和乙醇脱氢酶的基因,植物乳杆菌的胞外多糖还具有药物和保健作用。但是现有的植物乳杆菌最适pH偏中性,饮用过程中,在经过胃时极容易灭活。
发明内容
本发明目的在于针对现有技术的上述不足,提供一种高表达乙醛脱氢酶的耐酸植物乳杆菌及该菌发酵酸酸乳的制备方法和应用。
本发明的目的可通过以下技术方案实现:
一种植物乳杆菌(Lactobacillus plantarum)YLA1,所述植物乳杆菌保藏于中国典型培养物保藏中心,保藏日期为2020年7月6日,保藏号为CCTCC NO:M2020289。
该植物乳杆菌通过如下方法分离筛选得到:
1、选择合适的发酵泡菜样本,通过MRS和M17培养基,稀释涂布分离筛选出长势良好的各类细菌,经反复验纯后,共获得19株生长良好的乳酸菌菌株,经添加多批次浓度梯度乙醛压力反复筛选后,获得1株生长较好的耐乙醛压力的菌株,并对其进行细菌16S rDNA基因PCR扩增,并测序鉴定,鉴定其为植物乳杆菌,命名为植物乳杆菌YLA1。
2、采用甘油冻存方法对纯化得到的菌株进行保存。
本发明所述的植物乳杆菌YLA1在制备发酵乳制品中的应用;优选在制备酸奶或酸酸乳中的应用。所述应用包括饮酒前后服用上述酸酸乳,用于缓解饮酒后因乙醛积累造成的脸红不退、心率加速、胃部不适及头疼的酒后症状现象。
本发明所述的植物乳杆菌YLA1制备的酸奶或酸酸乳。
作为本发明的一种优选,所述的酸奶或酸酸乳中包括如下质量百分数的组分:奶粉8~11.5%、蔗糖4~7%、植物乳杆菌YLA1菌悬液2%~5%。
作为本发明的进一步优选,所述的酸奶或酸酸乳中活菌数为1.0×107~1.0×109CFU/g。
作为本发明的一种优选,所述的酸奶包含如下质量体积百分数的组分:奶粉8%~11.5%、蔗糖4%~7%、植物乳杆菌菌悬液(OD600≈1.5)2%~5%,德氏乳杆菌及嗜热链球菌复合菌悬液(OD600≈1.5)2%~5%,德氏乳杆菌及嗜热链球菌购自安琪酵母股份有限公司。
作为本发明的进一步优选,所述的酸奶或酸酸乳中活菌数为1.0×107~1.0×109CFU/g。
本发明所述的植物乳杆菌YLA1在发酵制备耐低pH的乙醛脱氢酶中的应用,所述的低pH为pH大于等于2.5。
本发明所述的植物乳杆菌YLA1在制备缓解因乙醛引起人的不适反应的益生菌产品,或用于制备缓解乙醛引起的不适反应的益生菌产品。
有益效果
本发明经稀释涂布、乙醛压力筛选等方法成功纯化得到一株可高表达乙醛脱氢酶的植物乳杆菌。其生长过程中乙醛脱氢酶基因可以大量转录表达,其发酵液具有高效的乙醛脱氢酶活性。上述植物乳杆菌既具有高效乙醛脱氢酶活性,又具备乳酸菌耐酸的特性,可有效防止胃酸对其的破坏,能够发挥较长时间的乙醛脱氢酶活性,缓解乙醛刺激导致的脸红不退、心率加速、胃部不适及头疼的酒后症状,可用于增加人体对酒精的耐受力,增进酒精在人体中的代谢速率,具有较好的实际应用价值,可应用于食品,药品等领域解酒产品的开发。
附图说明
图1植物乳杆菌YLA1菌体形态
图2植物乳杆菌YLA1系统进化发育树
图3乙醛脱氢酶aldH PCR扩增产物电泳结果
M:DL5000Marker泳道A、B:乙醛脱氢酶aldH扩增产物
图4普通植物乳杆菌、YLA1细胞破碎液以及体外表达ALDH酶的电泳。
泳道:1.Marker;2.普通植物乳杆菌破碎液;3.YLA1破碎液;4.利用表达载体体外表达的ALDH。
图5YLA1菌株对pH值耐受
生物材料保藏证明
Lactobacillus plantarum YLA1,保藏于中国典型培养物保藏中心,保藏地址中国武汉武汉大学,保藏日期为2020年7月6日,保藏号为CCTCC NO:M2020289。
具体实施方式
下面结合具体实施例对本发明做进一步阐释。
实施例1:
植物乳杆菌的分离鉴定过程如下:
选择合适的发酵泡菜样本,通过MRS和M17培养基,稀释涂布分离筛选出长势良好的各类细菌,经反复验纯后,共获得19株生长良好的乳酸菌株,经添加多批次浓度梯度乙醛压力反复筛选后,获得1株生长较好的耐乙醛压力的菌株,并对其进行细菌16S rDNA基因PCR扩增,并测序鉴定,鉴定其为植物乳杆菌,命名为植物乳杆菌YLA1
植物乳杆菌培养条件摸索及驯化
一、培养条件的摸索
采用MRS培养基设立6组培养条件:1)在37℃下,离心管中震荡培养;2)在37℃下,离心管中静置培养;3)在30℃下,离心管中静置培养;4)在37℃下,试管中震荡培养;5)37℃下,试管中静置培养;6)在30℃下,试管中静置培养。
最终确定最优培养条件为:在37℃下,试管静置培养。
二、设立梯度驯化乙醛浓度
液体37℃静置培养:MRS培养基中添加0.1%葡萄糖及不同浓度的乙醛;
固体37℃静置培养:上述液体培养基中添加2%琼脂;
驯化步骤:A浓度液体培养→A浓度固体培养→从A浓度固体上取菌落在下一个B浓度液体中培养→B浓度固体培养(以此类推),所有浓度实验均设置不含乙醛的对照组。
初步驯化:
固体培养也加入相应浓度乙醛,乙醛母液:孔径0.22μm的滤器滤过的40%(V/V)乙醛。
再次驯化:
以下实验液体培养16h,固体培养36h。
浓度2000μg/mL液体培养20h,固体培养48h;
浓度3000μg/mL、4000μg/mL液体培养22h,固体培养48h。
经梯度驯化后,挑取在高浓度乙醛平板上长势良好的菌株,进一步鉴定;
实施例2:
形态学特征
在乙醛浓度4000μg/mL的平板上挑取4个菌落PCR扩增16S rRNA基因,最终选取两个测序。经NCBI比对其核酸序列,获得与其相似性较高的细菌的16S rRNA基因序列,进一步将其与同源性较高的100株细菌的16S rRNA基因进行序列比对,采用Neighbor-Join法构建系统进化发育树(图2),结果发现其与植物乳杆菌JCM1149的相似性为99.87%,鉴定为植物乳杆菌。将其交予武汉大学的中国典型培养物保藏中心保藏,保藏日期为2020年7月6日,保藏号为CCTCC NO:M2020289.
植物乳杆菌YLA1乙醛脱氢酶基因(aldH)的PCR扩增与鉴定:
植物乳杆菌YLA1(Lactobacillus plantarum)aldH的鉴定
1、YLA1基因组抽提及aldH电泳检测
a.按照生工公司的SanPrep柱式质粒基因组小量抽提试剂盒说明书进行实验,基因组于-20℃保存。
b.根据Lactobacillus plantarum DSM16365基因序列,设计引物扩增aldH序列。引物如下:
aldH-f:5’ATGCTGAAAGAAATGGAAGA 3’
aldH-r:5’TTAGCGAATCTTAGTTAAGC 3’
aldH基因PCR体系遵循下表中各个体积添加到已灭菌的PCR管中进行扩增。
PCR反应体系
程序:98℃4min,98℃10s,56℃15s,72℃30s,32个循环,72℃再延伸10min,4℃保存。
凝胶电泳检测。吸取将上述PCR产物分装5管,每管加入DNA-Loading buffer,后将样品点样于点样孔中,并在电压为100V,时间40min左右电泳结束,凝胶成像仪观察条带大小,结果如图3所示,条带单一,大小1400bp左右,与预期大小一致。测序结果显示YLA1aldH基因序列如SEQ ID NO.1所示。
实施例3:植物乳杆菌YLA1乙醛脱氢酶的检测及与其他菌株的比较:
体外测定乙醛脱氢酶活的方法(Solarbio乙醛脱氢酶活性检测试剂盒):
取YLA1菌株(实验组)、普通植物乳杆菌菌株(对照1)、大肠杆菌菌株(对照2)分别按5%的接种量接入2‰(v/v)乙醛的MRS培养基中,37℃静置培养12h,离心取菌体,用PBS(7.4)缓冲液洗涤菌体2次,再用提取液重悬菌体,冰浴超声破碎(功率300w,超声3秒,间隔7秒,总时间30min);然后1000g,4℃,离心20min去上清进行酶活力测定。各试剂的组分参照Solarbio乙醛脱氢酶活性检测试剂盒说明书,测定体系如下:
酶活测定反应体系
按上述反应体系测乙醛脱氢酶酶活,总体系200μL,将上述试剂充分混匀后加样于96孔板,于OD340nm处测定30s吸光值A1;37℃分别孵育90s,5min,10min,15min,20min,25min,30min后于OD340nm处测定吸光值A2。
计算ΔA测定管=A2测定-A1测定,ΔA空白管=A2空白管-A1空白管,ΔA=ΔA测定管-ΔA空白管。酶活定义:每mL样本每分钟生成1nmol的NADH定义为一个酶活单位。
ALDH酶活(U/mL)=ΔA÷(ε×d)×109×V反总÷V样÷T=804×0.6×ΔA
ε:NADH摩尔消光系数,6.22×103L/mol/cm;;d:96孔板光径,0.6cm;V反总:反应体系总体积,2×10-4L;V样;反应体系中样本体积,0.04mL;T:反应时间,1min;109:单位换算系数,1mol=109nmol.
实验结果:
每孔均上样10微升进行电泳,普通植物乳杆菌、YLA1细胞破碎液以及体外表达ALDH酶的电泳图见图4,可见菌株YLA1细胞破碎液中ALDH酶的含量高于普通植物乳杆菌。
2.乙醛脱氢酶活性检测试剂盒检测结果:
酶活检测结果可见,YLA1菌株的乙醛脱氢酶酶活远高于普通植物乳杆菌(对照一),大肠杆菌(对照二)基本无此酶活。
实施例4YLA1菌株对pH值耐受的测定
方法:将YLA1按照2%的接种量接入pH分别为1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5、5.0的MRS肉汤培养基中,37℃静置培养30min后取10μL点在MRS固体培养基上,观察细菌生长情况。
实验结果:YLA1可以耐受的pH值为大于等于2.5。
实施例5
1.单菌发酵酸酸乳方案
采用MRS培养基在37℃下,试管静置培养制备植物乳杆菌YLA1菌悬液100mL,5000g离心收菌。PBS缓冲液稀释至107CFU/mL。
发酵乳的制备:
制备的100mL全脂复原乳,方法如下,称量1.5g全脂奶粉和6.5g蔗糖,先用适量白开水溶解,再定容至100mL,并加热到60℃,放入65℃水浴锅进行巴氏杀菌30min。按照总接种量2×105CFU/mL接种,在37℃恒温箱中发酵,当pH值降至5.0时,终止发酵并置于4℃冰箱中。
2.混菌发酵酸奶方案
德氏乳杆菌、嗜热链球菌和植物乳杆菌YLA1不同比例复配发酵酸奶
制备植物乳杆菌YLA1和德氏乳杆菌、嗜热链球菌复合菌悬液100mL,所含德氏乳杆菌、嗜热链球菌的比例为1:1,5000-6000g离心收菌。PBS缓冲液稀释至107CFU/mL。
发酵乳的制备:
称量1.5g全脂奶粉和6.5g蔗糖,先用适量白开水溶解,再定容至100mL,并将其加热到60℃,放入65℃水浴锅进行巴氏杀菌30min。按照总接种量2×105CFU/mL接种。接种后于42℃水浴锅中升温,后置于37℃培养箱中发酵。当pH降至4.5时放冰箱冷却。
嗜热链球菌和植物乳杆菌YLA1加入比例如下表所示。
表1. 1德氏乳杆菌、嗜热链球菌复合菌和植物乳杆菌加入比例
发酵产物活菌数的测定(总活菌)
发酵好的酸奶至于4℃冰箱保存,每48h取酸奶用PBS缓冲液(pH 7.2~7.4)稀释至一定倍数后,涂布MRS琼脂培养基平板,置于在37℃培养48h进行菌落计数。
4℃冰箱保存20天,保存前酸奶中活菌数为2.6×107CFU/mL,20天后酸奶中活菌数仍能保持在1.6×107CFU/mL。
序列表
<110> 安徽农业大学
<120> 一种高表达乙醛脱氢酶的耐酸植物乳杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1368
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum)
<400> 1
atgctgaaag aaatggaaga aacaaccgta tcacgttcaa tcgatcggtt agtcttaaat 60
gcttcgttag ctgccaaccg tcttgaagtc atggaccaaa gtcaagttga tcaggctgtc 120
gctgccatgg cccgcgctgc ccacgctgct cgtggcatgc tggccgctat ggccgtcgaa 180
gaaacgggtc gcggaaatta tcgtgataaa gttgcgaaga acgactttgc agccaaaaac 240
gtttataact acatcaagga tgacaagacg gtcggtatca ttaatgacga tccagtcagt 300
ggcgtgatga aagttgctga accagttgga attattgcgg gggtcacccc agttaccaac 360
ccaacatcaa ccgtcatttt caatgccatg ttagcattaa agactcgcaa tcccattatt 420
tttggtttcc atccctttgc acaaaaatct tgtgttgaaa ctggccgaat catccgcgat 480
gctgctattg ccgctggcgc tcctaaggat tggattcagt ggatcaagac gcctagcctt 540
gaagcaacca acaccttgat gaaccatccg ggcgtcgcta ccattattgc aactggcggt 600
gccggcatgg tcaagaccgc gtattcaact ggtaaaccgg cactcggtgt tggccctggt 660
aacgtgccat gcttcatcga gcaaaccgca gacattcaac aggcagtcag tgatgtcgtc 720
acttccaagt cattcgacaa cggcatgatc tgtgcttccg aatcaaactt aatcgttgct 780
gatcaaatct atgatcaagt taaacgtgaa ttaagtcaca acggtgtgta ctttgtcggt 840
accgagaact tcaaggcctt agaagcaact gtcatgaacc tggataaaca ggctgttgac 900
ccgaaagtag ctgggcaaac gccatggcaa atcgctcagt gggctggctt tgatgtccca 960
tccgatacca aagtattagc agttgagttg cctagcatcg gtggtgacca agtcttatca 1020
cgagaaaagt tatcaccagt cctcgccgtc gttcatgcca aggatactga ggccggcttc 1080
aacctgatga aacgcagcct agcacttggc ggactgggac atacggccgc cttgcatacg 1140
actgacgaag ctgttatgaa caagtttgcc ttagaaatga ctgcttgtcg agcattgatc 1200
aacgtgccgt cttcacaagg tgccattggt tataaatatg ataacgtcgc accatcctta 1260
acactcggtt gtggaacatg ggggcataac tcgatttcac acaacttgga agattgggat 1320
ctattaaata ttaagaccgt tgcaaaacgc ttaactaaga ttcgctaa 1368
Claims (6)
1.一种植物乳杆菌(Lactobacillus plantarum)YLA1, 所述植物乳杆菌保藏于中国典型培养物保藏中心,保藏日期为2020年7月6日,保藏号为CCTCC NO:M2020289。
2.权利要求1所述的植物乳杆菌YLA1在制备发酵乳制品中的应用。
3.根据权利要求2所述的应用,其特征在于,权利要求1所述的植物乳杆菌YLA1在制备酸奶或酸酸乳中的应用。
4.权利要求1所述的植物乳杆菌YLA1制备的酸奶或酸酸乳,其特征在于所述的植物乳杆菌YLA1为活菌。
5.根据权利要求4所述的酸奶或酸酸乳,其特征在于所述的酸奶或酸酸乳中活菌数为1.0×107~1 .0×109 CFU/g。
6.权利要求1所述的植物乳杆菌YLA1在制备缓解因乙醛引起人的不适反应的益生菌产品,或用于制备缓解乙醛引起的不适反应的益生菌产品。
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