CN112251383A - 一株产苯乳酸的干酪乳杆菌及其应用 - Google Patents
一株产苯乳酸的干酪乳杆菌及其应用 Download PDFInfo
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- CN112251383A CN112251383A CN202011174207.2A CN202011174207A CN112251383A CN 112251383 A CN112251383 A CN 112251383A CN 202011174207 A CN202011174207 A CN 202011174207A CN 112251383 A CN112251383 A CN 112251383A
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- China
- Prior art keywords
- lactobacillus casei
- vinegar
- phenyllactic acid
- bran
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Links
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- C—CHEMISTRY; METALLURGY
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- C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
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Abstract
本发明涉及微生物技术领域，具体涉及一株从复杂体系四川食醋醋醅中分离筛选出的产苯乳酸的干酪乳杆菌。该产苯乳酸的干酪乳杆菌Lactobacillus casei菌株号为R2‑8，在中国微生物菌种保藏管理委员会普通微生物中心登记保藏；保藏号为CGMCC No.20579。接种干酪乳杆菌Lactobacillus casei R2‑8麸醋醋醅中苯乳酸产量为2.1 g/L，本发明提供的产苯乳酸干酪乳杆菌R2‑8应用于四川麸醋醋醅中，可显著提高醋醅中风味物质的种类和含量，可为我国麸醋新功能的开发提供理论依据。
Description
技术领域
本发明涉及微生物技术领域，具体涉及一株从四川食醋醋醅中分离筛选出产苯乳酸的干酪乳杆菌，其能应用于食醋生产发酵。
背景技术
苯乳酸（phenyllactic acid, PLA），别名3-苯基乳酸，是一种安全可靠、高效的抑菌物质，具有较为广泛的抑菌谱，较低浓度的苯乳酸能有效抑制细菌、真菌等大部分食品腐败菌，其效果比一些常见的食品防腐剂显著，如苯甲酸钠、三梨酸钾，此外，苯乳酸还具有易溶、易扩散和稳定性高等优点，可广泛用于食品、饮料、医药和化妆品行业中，具有广阔的应用前景。
目前，已知苯乳酸的制备方法有化学合成和微生物合成法。但由于化学合成过程繁琐，且有较多的污染性副产物，因此，微生物合成苯乳酸成为众多研究者的选择。目前，已发现有多种微生物可合成苯乳酸。例如白地霉（Geotrichum candidum）、丙酸菌(Propioniacterium)、片球菌（Pediococcus）、红球菌（Rhodococcus）和乳酸菌（Lactobacillus）等，其中产苯乳酸的微生物以乳酸菌居多，特别是植物乳杆菌。2000年，首次报道从酸面团中分离出一株可产生56 mg/L苯乳酸的植物乳杆菌21B。李士龙从自然发酵食品筛选到一株植物乳杆菌LY-78，在培养基中苯乳酸的产量为0.246 g/L；黄国昌对植物乳杆菌BLPC002发酵培养基优化后，其可产生1.67 g/L的苯乳酸；关今韬从泡菜中分离到了一株布氏乳杆菌GBS3，在优化培养基后可产生苯乳酸10.93 g/L。吴仁蔚等检测了77种食醋中苯乳酸的含量，发现谷物酿造食醋中苯乳酸含量显著果醋，最高含量为979mg/L。目前，这些产苯乳酸的乳酸菌的研究主要针对其产苯乳酸的能力以及其抑菌作用，而对菌株应用于生产发酵的实例较少，2020年，J.-J.XU等人优化L. crustorum NWAFU 1078的发酵条件后，将其应用于分批发酵与连续发酵，发现该菌株在两个发酵系统中能分别产生15.2 mmol/L苯乳酸（苯丙酮酸的转化率为76%）和6.5 mmol/L苯乳酸（苯丙酮酸的转化率为55%），具有较高的苯乳酸产量。但未见源于食醋分离的产苯乳酸的干酪乳杆菌应用于麸醋发酵的研究报道。本专利中产苯乳酸干酪乳杆菌筛选自复杂体系食醋醋醅，同时将其应用于麸醋发酵，结果表明干酪乳杆菌R2-8产苯乳酸的同时，可显著提高醋醅中风味物质的种类和含量，充分说明干酪乳杆菌R2-8在食醋发酵中具有独特的应用潜力，可为我国食醋新功能的开发提供理论依据。
发明内容
针对现有技术存在的问题，本发明目的在于提供一株高产苯乳酸的干酪乳杆菌Lactobacillus casei R2-8。
本发明所提供的菌株为干酪乳杆菌Lactobacillus casei R2-8，保藏于中国普通微生物菌种保藏管理中心，保藏地点为北京市朝阳区北辰西路1号院，保藏号为CGMCC No.20579，保藏日期为2020年08月28日。
本发明提供高产苯乳酸的乳酸菌菌株的分离筛选、鉴定、目的菌株的生长特性及其在模拟食醋生产发酵时应用于麸醋醋醅中的研究。
取1mL特级生醋，于100mL无菌MRS液体培养基中，37℃培养24 h；在无菌条件下称取25 g醋醅，于225 mL无菌生理盐水中，分别取培养后的生醋增殖液和生理盐水稀释的醋醅液各1 mL，用无菌生理盐水进行10倍梯度稀释，取适宜稀释度样液200μL涂布于含CaCO3的MRS固体培养基平板，37℃培养24~48 h，挑取不同菌落形态、不同生长位置、溶钙圈明显的单菌落进行多次平板划线获得纯培养物，保存备用。
将上述获得的乳酸菌进行高产苯乳酸菌株的初筛，将上述筛选的菌株划线接种于MRS固体培养基平板，划线长度约为2 cm，37℃培养48 h。取20 µL已活化好的大肠杆菌和金黄色葡萄球菌菌体悬浮液于半固体LB培养基中混匀后，取10 mL于乳酸菌划线的MRS固体培养基表面，凝固后，37℃培养16 h，观察抑菌情况。
高产苯乳酸乳酸菌菌株的复筛，挑取上述抑菌圈较大的单菌落，于MRS液体培养基，37℃培养48 h。取10 mL菌液于离心管中，再置于80℃水浴加热30 min，4℃ 8000 r/min离心15 min，取上清液用高效液相色谱法测定液体中苯乳酸的浓度。
菌株形态鉴定：肉眼观察菌落形态为圆形，凸起，边缘整齐，表面湿润的乳白色菌落，在含CaCO3的MRS固体平板上形成溶钙圈；光学显微镜下菌株细胞呈杆状，革兰氏染色阳性；经乳酸菌生化鉴定结果表明，该菌株可利用菊糖、甘露醇、水杨苷、纤维二糖、山梨醇、蔗糖、乳糖，不利用麦芽糖、七叶苷、棉子糖。根据《伯杰氏细菌鉴定手册》和《乳酸细菌分类鉴定及实验方法》，结合该菌株的菌落和细胞形态特征以及乳酸菌生理生化鉴定结果，初步判定菌株R2-8为乳杆菌属。
菌株R2-8分子遗传学鉴定。
菌株R2-8经16S rDNA序列测定，获得如下序列：
TCGCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTCAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTATCATGGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTTTAAGTTGAATCACAATGCAAGCACCGTTCATCGACCAAAACTCGTTCGACTTGCATTATAGCACCCGCCG
该菌株的16S rDNA基因测序结果与GenBank 数据库中序列进行Blast同源性对比，该菌株序列与Lactobacillus casei菌株基因序列高度同源，序列提交NCBI获得登录号为MT998495，结合形态学和生理生化指标，最终将菌株R2-8鉴定为干酪乳杆菌。
本发明所提供的高产苯乳酸的菌株是从四川食醋醋醅中分离筛选得到的，该菌株在生长pH值为7.0、培养温度为37℃条件下培养能代谢合成2.0 g/L的苯乳酸。
本发明所用培养基为MRS液体培养基：蛋白胨10 g，牛肉膏10 g，酵母粉5 g，磷酸氢二钾2 g，柠檬酸氢二铵2 g，乙酸钠5 g，葡萄糖20 g，吐温-80 2 g，七水硫酸镁0.58 g，四水硫酸锰0.25 g，2.0 g苯丙氨酸，水1000 mL，调pH至6.2±0.2，121℃灭菌15 min；加入2%琼脂粉、1.5% CaCO3为MRS固体培养基。
与现有技术相比，本发明的积极效果是：首次从复杂体系四川酿造食醋醋醅中筛选获得产苯乳酸的干酪乳杆菌，且该菌株代谢产生的苯乳酸含量为2.0 g/L；将该菌株应用于食醋发酵生产的醋醅中，提高了醋醅中的苯乳酸含量、挥发性组分种类和游离氨基酸总量。表明干酪乳杆菌R2-8能提高食醋的品质，充分说明干酪乳杆菌R2-8在食醋发酵中具有独特的应用潜力，可为我国食醋新功能的开发提供理论依据。
附图说明
图1为高产苯乳酸R2-8的菌落形态图和细胞形态图。
图2为高产苯乳酸R2-8菌株的进化树。
图3为高产苯乳酸R2-8菌株的生长曲线。
图4为四川麸醋固态发酵工艺流程图
具体实施方式
实例1：菌株 R2-8的生长特性。
干酪乳杆菌R2-8的生长曲线将活化3次后的R2-8菌株以1%(v/v)接种于MRS液体培养基中，于37℃下恒温静置培养，分别于0 h、4 h、8 h、12 h、16 h、20 h、24 h、30 h、36 h、42 h、48 h、72 h、96 h取出培养物，在600 nm波长下测定吸光度。以所测OD值为纵坐标，培养时间为横坐标，绘制该干酪乳杆菌菌株的生长曲线。
初始pH值对R2-8菌株生长的影响将活化3次后的R2-8菌株以1%(v/v)接种于初始pH值分别为5.5、6.0、6.5、7.0、7.5的MRS液体培养基中，37℃恒温静置培养12 h后测定菌悬液在600 nm处的OD值。在以上实验pH值范围内，随着初始pH值的增加，菌株生长状态越好，其最适生长初始pH值7.0。
实例2：菌株R2-8应用于四川麸醋模拟发酵体系
（1）干酪乳杆菌R2-8种子液的制备：将甘油保藏的干酪乳杆菌R2-8于MRS液体培养基中活化（37℃，24±2 h），按2%的接种量接种于装有100 mL基础培养基的300 mL三角瓶中，37℃培养24±2 h，即为干酪乳杆菌种子液。
（2）按工艺配方（四川某麸醋公司提供）称取麸皮、小麦、醋糟混合搅拌2 min，使之充分拌匀，控制搅拌后的物料水分在55%-58%，将拌好的物料放入1000 mL三角瓶中。
（3）3 d后摇动装有发酵物的三角瓶，使醅料松散，同时加入10%活化好的干酪乳杆菌R2-8种子液，之后每隔2 d摇动一次三角瓶。以不加干酪乳杆菌种子液为对照组。控制发酵温度：0-3 d≤35℃，3-8d在35℃-42℃之间，9-15 d在35℃-40℃之间，发酵结束即为成熟醋醅。
（4）采用高效液相色谱仪、气相色谱质谱联用仪和氨基酸自动分析仪分别对实验组与对照组中成熟醋醅的苯乳酸含量、挥发性香气物质和游离氨基酸总量进行测定，分析得出试验组中检测出34种挥发性组分，高于对照组中21中挥发性组分；且苯乳酸和游离氨基酸总量分别为2.1 g/L、1301mg/100 mL和1.4 g/L、902 mg/100 mL，结果表明干酪乳杆菌Lactobacillus casei R2-8提高了四川麸醋醋醅的风味品质，其能应用于四川麸醋醋醅发酵，对提升食醋品质具有潜在价值。
以上的实例仅仅是对本发明的实施方式进行描述，而并非对本发明的范围进行限定，对于本领域的技术人员来说，可以对上述说明进行改进或变形，但所有的这些改进或变形均应落入本发明的权利要求确定的保护范围。
序列表
<110> 四川农业大学
<120> 一株产苯乳酸的干酪乳杆菌及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1462
<212> DNA
<213> 干酪乳杆菌(Lactobacillus casei)
<400> 1
tcgcttagac ggctcgctcc ctaaaagggt tacgccaccg gcttcgggtg ttacaaactc 60
tcatggtgtg acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcgtgctgat 120
ccgcgattac tagcgattcc gacttcgtgt aggcgagttg cagcctacag tccgaactga 180
gaatggcttt aagagattag cttgacctcg cggtctcgca actcgttgta ccatccattg 240
tagcacgtgt gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc 300
tccggtttgt caccggcagt cttactagag tgcccaactc aatgctggca actagtcata 360
agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca 420
tgcaccacct gtcattttgc ccccgaaggg gaaacctgat ctctcaggtg atcaaaagat 480
gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct ccaccgcttg 540
tgcgggcccc cgtcaattcc tttgagtttc aaccttgcgg tcgtactccc caggcggaat 600
gcttaatgcg ttagctgcgg cactgaaggg cggaaaccct ccaacaccta gcattcatcg 660
tttacggcat ggactaccag ggtatctaat cctgttcgct acccatgctt tcgagcctca 720
gcgtcagtta cagaccagac agccgccttc ggccactggt gttcttccat atatctacgc 780
atttcaccgc tacacatgga gttccactgt cctcttctgc actcaagttt cccagtttcc 840
gatgcgcttc ctcggttaag ccgagggctt tcacatcaga cttaaaaaac cgcctgcgct 900
cgctttacgc ccaataaatc cggataacgc ttgccaccta cgtattaccg cggctgctgg 960
cacgtagtta gccgtggctt tctggttgga taccgtcacg ccgacaacag ttactctgcc 1020
gaccattctt ctccaacaac agagttttac gacccgaaag ccttcttcac tcacgcggcg 1080
ttgctccatc agacttgcgt ccattgtgga agattcccta ctgctgcctc ccgtaggagt 1140
ttgggccgtg tctcagtccc aatgtggccg atcaacctct cagttcggct acgtatcatg 1200
gccttggtga gccgttacct caccaactag ctaatacgcc gcgggtccat ccaaaagcga 1260
tagcttacgc catctttcag ccaagaacca tgcggttctt ggatttatgc ggtattagca 1320
tctgtttcca aatgttatcc cccacttaag ggcaggttac ccacgtgtta ctcacccgtc 1380
cgccactcgt tttaagttga atcacaatgc aagcaccgtt catcgaccaa aactcgttcg 1440
acttgcatta tagcacccgc cg 1462
Claims (3)
1.一株产苯乳酸的干酪乳杆菌 Lactobacillus casei R2-8，其特征在于保藏于中国普通微生物菌种保藏管理中心，保藏地点为北京市朝阳区北辰西路1号院，保藏号为CGMCCNo. 20579，保藏日期为2020年08月28日。
2.根据权利要求1所述干酪乳杆菌Lactobacillus casei R2-8的应用，其特征在于：干酪乳杆菌Lactobacillus casei R2-8分离自复杂体系四川酿造食醋醋醅，并应用于麸醋醋醅发酵。
3.根据权利要求2所述干酪乳杆菌Lactobacillus casei R2-8应用于麸醋醋醅发酵，其特征在于：将干酪乳杆菌Lactobacillus casei R2-8种子液以10%接种量接种于以麸皮、小麦及酒糟为醅料的醋醅中，发酵制得成熟醋醅（试验组），以不接种干酪乳杆菌Lactobacillus casei R2-8为对照组，分析检测得出试验组中检测出34种挥发性组分，而对照组只检测出21种挥发性组分，苯乳酸和游离氨基酸总量分别为2.1 g/L、1301 mg/100mL和1.4 g/L、902 mg/100 mL，干酪乳杆菌Lactobacillus casei R2-8能提高四川麸醋醋醅的风味品质，具有潜在应用价值。
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| CN113150941A (zh) * | 2021-05-27 | 2021-07-23 | 华中农业大学 | 一种富含苯乳酸的食醋、制备方法及应用 |
| CN115074290A (zh) * | 2022-07-06 | 2022-09-20 | 福建生物工程职业技术学院 | 一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌及其应用 |
| CN117431189A (zh) * | 2023-12-08 | 2024-01-23 | 四川吉恒食品有限公司 | 一株副干酪乳杆菌副干酪亚种菌株qh-20029及其应用 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113150941A (zh) * | 2021-05-27 | 2021-07-23 | 华中农业大学 | 一种富含苯乳酸的食醋、制备方法及应用 |
| CN115074290A (zh) * | 2022-07-06 | 2022-09-20 | 福建生物工程职业技术学院 | 一种联产苯乳酸和γ-氨基丁酸的干酪乳杆菌及其应用 |
| CN117431189A (zh) * | 2023-12-08 | 2024-01-23 | 四川吉恒食品有限公司 | 一株副干酪乳杆菌副干酪亚种菌株qh-20029及其应用 |
| CN117431189B (zh) * | 2023-12-08 | 2024-03-15 | 千禾味业食品股份有限公司 | 一株副干酪乳杆菌副干酪亚种菌株qh-20029及其应用 |
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