CN112501063B - 一株高产gaba的植物乳杆菌lzu-j-tsl6及其应用 - Google Patents
一株高产gaba的植物乳杆菌lzu-j-tsl6及其应用 Download PDFInfo
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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Abstract
本发明属于微生物技术领域,具体涉及一株高产GABA的植物乳杆菌LZU‑J‑TSL6及其应用。本发明提供了一株高产GABA的植物乳杆菌Lactobacillus plantarum LZU‑J‑TSL6,所述植物乳杆菌Lactobacillus plantarum LZU‑J‑TSL6于2020年10月23日保藏于广东省微生物菌种保藏中心,保藏编号为:GDMCC No:61242。本发明提供的植物乳杆菌LZU‑J‑TSL6具有高产γ‑氨基丁酸(GABA)的能力,产量高于3.838g/L;并且所述植物乳杆菌LZU‑J‑TSL6具有一定的抑菌活性,能够显著抑制金黄色葡萄球菌的活性;同时植物乳杆菌LZU‑J‑TSL6具有较强的胃肠液耐受性,在模拟胃液和肠液中培养后的存活率显著高于鼠李糖乳杆菌LGG,可在胃肠道定植。
Description
技术领域
本发明属于微生物技术领域,具体涉及一株高产GABA的植物乳杆菌LZU-J-TSL6及其应用。
背景技术
γ-氨基丁酸又名4-氨基丁酸(4-Aminobutanoicacid,4-AB,简称GABA)和γ-氨酪酸,广泛存在于自然界,是一种非蛋白质组成的天然氨基酸,由谷氨酸(Glutamic acid,Glu)经谷氨酸脱羧酶催化而来,以自由态形式广泛存在于原核生物和真核生物中。GABA具有许多重要的生理功能,如降低血压、镇静、增强记忆、调节呼吸系统、抗焦虑,尤其对更年期的失眠、压抑和自身失调疗效良好。此外,GABA还具有改善人体肝功能,活化肾功能,预防肥胖,防止皮肤老化,促进乙醇代谢,消臭作用等功效。随着GABA的生理功能不断研究和阐明,已经发展成为一种新型的功能性因子,在功能性食品的开发中有很大潜力,正逐渐应用于医药、食品等行业。
目前对于GABA的制备方法主要包括有化学合成法和生物合成法。其中,化学合成法较为常见,主要方法为:①将邻苯二甲酰亚胺钾和4-氯丁氰进行反应,产物经过浓硫酸回流水解后结晶提纯得到;②以吡咯烷酮作为原料,经过氢氧化钙和碳酸氢铵水解开环制得GABA。虽然化学合成方法来制备GABA具有反应速度快的优势,但由于其合成成本较高,而产率较低,并且在其生产工艺中使用了危险溶剂、甚至有毒溶剂,因而化学合成法制备的GABA不能用于食品,也不能被认为是天然的食品添加剂。
微生物转化法主要是以谷氨酸钠或谷氨酸为底物,利用微生物的活性,将底物转化为γ-氨基丁酸,例如中国专利CN103320362A公开了一种利用植物乳杆菌(Lactobacillus plantarum)SK30.001以谷氨酸钠为底物转化生产γ-氨基丁酸的方法,但是γ-氨基丁酸产量低、转化时间长、转化率低等缺陷,效果不太理想;而中国专利CN105087699A公开了一种利用短乳杆菌(CGMCC NO:3414)转化L-谷氨酸钠和谷氨酸的混合底物催化转化合成γ-氨基丁酸的方法,上述方法需要额外加入大量的L-谷氨酸钠和谷氨酸为底物,成本较高;中国专利CN108467860A公开了一种通过蛋白质工程改造巨大芽孢杆菌来源的谷氨酸脱羧酶,改造后的谷氨酸脱羧酶基因连接于pET24a载体,在E.coli BL21(DE3)中表达,并在发酵罐中培养重组菌株,用于转化谷氨酸生产γ-氨基丁酸;利用基因工程并通过大肠杆菌重组表达,生物转化生产GABA,虽然获得的产量较高,但是基因工程改造过程复杂,成本较高。
直接利用微生物发酵生产GABA具有节约生产成本、缩短生产周期及降低环境污染等诸多优点。但是大部分微生物的胃肠耐受性差,不能在胃肠道中定植,一般不能直接作为食品或食品添加剂使用。
针对上述技术问题,本发明从发酵食品中分离获得了一株植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6,所述植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6具有高产γ-氨基丁酸(GABA)的能力,产量高达3.838g/L;并且所述植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6具有一定的抑菌活性,能够显著抑制金黄色葡萄球菌的活性;同时植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6具有较强的胃肠液耐受性,在模拟胃液和肠液中培养后的存活率显著高于鼠李糖乳杆菌LGG,可在胃肠道定植。
发明内容
针对上述问题,本发明的目的在于提供一种植物乳杆菌(Lactobacillusplantarum)LZU-J-TSL6,所述植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6于2020年10月23日保藏于广东省微生物菌种保藏中心,保藏编号为:GDMCC No:61242,保藏地址为:广州市先烈中路100号大院59号楼5楼;联系电话:020-87137633。
本发明的另一目的在于提供一种植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6在生产γ-氨基丁酸中的应用。
本发明的另一目的在于提供一种植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6在制备抑制肠道致病菌药物中的应用。
优选地,所述肠道致病菌为金黄色葡萄球菌。
本发明的另一目的在于提供一种植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6在制备调节肠道菌群保健品中的应用。
本发明的另一目的在于提供一种利用植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6生产γ-氨基丁酸的方法,所述方法为:先将植物乳杆菌(Lactobacillusplantarum)LZU-J-TSL6接入种子培养基中进行种子培养,再接入发酵培养基中进行发酵培养;所述种子培养基为MRS液体培养基,所述发酵培养基为GYP液体培养基。
优选地,在种子培养前先对植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6进行活化,具体过程为:将植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6菌种接种到MRS固体培养基上,37℃,培养48h。
优选地,所述MRS固体培养基的组成为:蛋白胨10g/L,牛肉膏10g/L,酵母浸粉5g/L,葡萄糖20g/L,醋酸钠5g/L,吐温-80 1mL,柠檬酸氢二铵2g/L,磷酸氢二钾2g/L,七水硫酸镁0.58g/L,一水硫酸锰0.25g/L,碳酸钙4g/L,琼脂15g/L。
优选地,所述MRS液体培养基的组成为:蛋白胨10g/L,牛肉膏10g/L,酵母浸粉5g/L,葡萄糖20g/L,醋酸钠5g/L,吐温-80 1mL,柠檬酸氢二铵2g/L,磷酸氢二钾2g/L,七水硫酸镁0.58g/L,一水硫酸锰0.25g/L,碳酸钙4g/L。
优选地,所述GYP液体培养基组成为:葡萄糖13g/L,氯化钠0.001g/L,盐酸吡哆醇0.15g/L,四水硫酸锰0.001g/L,酵母浸膏5g/L,无水乙酸钠2g/L,七水硫酸亚铁0.001g/L,谷氨酸钠12g/L,七水硫酸镁0.02g/L。
优选地,所述种子培养条件为:25-40℃,培养7h以上。
优选地,所述种子培养条件为:37℃,培养16h。
优选地,所述发酵培养条件为:将种子培养的产物以高于2%的接种量接入所述发酵培养基中,25-40℃,培养12h以上。
优选地,所述发酵培养条件为:将种子培养的产物以高于4%的接种量接入所述发酵培养基中,37℃,静置培养48h。
附图说明
图1生产γ-氨基丁酸的植物乳杆菌的初步鉴定结果;
图2植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6的系统发育树;
图3植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6对模拟胃液的耐受性;
图4植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6对模拟肠液的耐受性;
图5植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6的抑菌结果。
具体实施方式
下面通过具体实施例对本发明进行详细的阐述,但本发明的保护范围并不限于以下实施例,任何本领域的技术人员在本发明的基础上,结合本领域公知常识所能想到的技术方案,都属于本发明的保护范围。
在本发明的下述实施例中所用菌株来源如下:
实验菌株:植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6于2020年10月23日保藏于广东省微生物菌种保藏中心,保藏编号为:GDMCC No:61242,并于2020年10月26日检测为存活状态;保藏地址为:广州市先烈中路100号大院59号楼5楼;联系电话:020-87137633。
鼠李糖乳杆菌LGG菌(菌种编号为BNCC 136673)、金黄色葡萄球菌(BNCC 186335)购自北纳创联生物技术有限公司。
MRS固体培养基:蛋白胨10g/L,牛肉膏10g/L,酵母浸粉5g/L,葡萄糖20g/L,醋酸钠5g/L,吐温-80 1mL,柠檬酸氢二铵2g/L,磷酸氢二钾2g/L,七水硫酸镁0.58g/L,一水硫酸锰0.25g/L,碳酸钙4g/L,琼脂15g/L;
MRS液体培养基:蛋白胨10g/L,牛肉膏10g/L,酵母浸粉5g/L,葡萄糖20g/L,醋酸钠5g/L,吐温-80 1mL,柠檬酸氢二铵2g/L,磷酸氢二钾2g/L,七水硫酸镁0.58g/L,一水硫酸锰0.25g/L,碳酸钙4g/L;
GYP液体培养基:葡萄糖13g/L,氯化钠0.001g/L,盐酸吡哆醇0.15g/L,四水硫酸锰0.001g/L,酵母浸膏5g/L,无水乙酸钠2g/L,七水硫酸亚铁0.001g/L,谷氨酸钠12g/L,七水硫酸镁0.02g/L。
其他实验材料和仪器如无特殊说明,均可通过商业途径购买。
实施例1菌株LZU-J-TSL6的分离与活性检测
1.1分离
将发酵乳制品取0.5g左右,用PBS分别稀释成10-1,10-2,10-3,10-4,10-5,10-6,10-7,10-8的浓度梯度,各个浓度吸100μL到加有碳酸钙的改良型MRS固体培养基上涂布均匀,每一浓度两个重复。放置于37℃恒温培养箱内培养48h后观察菌落以及透明圈产生的情况,便于进一步分离纯化。
1.2纯化培养
从以上培养的浓度梯度中选择一合适的梯度,挑选边界清晰可见并均有透明圈的单菌落15-25个,挑选出来的菌落采用划线法在新制的MRS固体培养基上传代培养24-48h,再次划线培养,此步骤共重复三次,以获得单克隆菌株。此菌株通过MRS液体培养基培养24h后,将菌液和50%甘油按1:1的比例混匀(甘油终浓度为25%),在-80℃保存。
1.3菌株活性筛选
取上述甘油保存的菌液按2%接种量接种到1.5mL的MRS液体培养基中并置于37℃恒温培养箱中培养10h作为种子菌液用于接下来的实验。
种子菌液按2%的接种量接种到2mL装有MRS液体培养基的EP管中,37℃条件下培养48h;挑取单菌落于MRS液体培养基上,37℃条件下培养16h;然后转入GYP液体培养基上,37℃,静置培养48h;取发酵液上清液1μL点样于滤纸上(每10样+1标/一张滤纸),2000rpm离心10min,在展开剂中层析4h后吹干滤纸,将8g/L的茚三酮喷到滤纸上,70℃烘箱中显色5min,初步筛选出具有产γ-氨基丁酸(GABA)的菌株共18株。
采用高效液相色谱法对18株菌株产GABA能力进行定量测定:
(1)标准溶液的配制:准确称取50.0mg的γ-氨基丁酸标准品于烧杯中,用双蒸水溶解,定容于50mL容量瓶中,摇匀,得到1.00mg/mL的标准储备液,4℃冰箱保存,备用;
(2)800μg/mg标准溶液配置:精确吸取3200μL标准储备液于5mL的EP管中,加800μL双蒸水,摇匀,得到800μg/mg标准溶液,4℃冰箱保存,备用;
(3)0.5mol/L硼酸钾溶液配置:称取3.1000g硼酸,2.6000g氢氧化钾于烧杯中,用双蒸水溶解,定容于100mL的容量瓶中,摇匀,用硝酸调节pH至9.5左右;
(4)衍生试剂/邻苯二甲醛(OPA)溶液配置:称取0.3000g的OPA,先溶解于5mL的甲醇,再溶解于100mL的硼酸钾溶液(0.5mol/L)中,然后加入0.25mL 2-巯基乙醇,混匀,避光4℃冰箱中保存(最多可保留1周,最好现配现用);
(5)磷酸二氢钾缓冲液的配制:称取1.0000g磷酸二氢钾于烧杯中,用双蒸水溶解,定容于100mL的容量瓶中,摇匀,得到0.01g/mL的磷酸二氢钾缓冲液,4℃冰箱保存,备用;
(6)醋酸-醋酸钠缓冲盐溶液(15mmol/mL)的配制:准确称取1.5830g的三水合乙酸钠于烧杯中,加入80μL冰醋酸,用双蒸水溶解,定容于1000mL容量瓶中,摇匀,过滤,备用。
色谱条件:色谱柱:Waters-C18色谱柱(250×4.6mm,5μm);流动相:醋酸-醋酸钠缓冲盐缓冲液(pH 5.80):甲醇=55:45;流速:0.8mL/min;柱温:30℃;检测波长:334nm;进样方式:手动进样;进样量:20μL。
实验结果如图1所示,分离获得的18株菌株均具有产GABA的能力,其中菌株LZU-J-TSL6产GABA能力最强,GABA产量高达3.838g/L。
实施例2菌株LZU-J-TSL6的鉴定与保藏
2.1菌株LZU-J-TSL6的16S rRNA的序列分析
提取菌株LZU-J-TSL6的DNA,使用细菌16S rRNA通用引物进行PCR扩增:
上游引物为:27F:AGAGTTTGATCMTGGCTCAG;
下游引物为:1492R:GGTTACCTTGTTACGACTT。
PCR扩增体系:50μL反应总体系,包含:2×Mix Taq,25μL;Primers(10μM):上游引物(Forward),2.0μL,下游引物(Reverse),2.0μL;DNA模板(100ng/μL),2.0μL;ddH2O,19μL。
PCR反应条件:94℃预变性,4min;94℃变性,1min;55℃复性,1min;72℃延伸,1.5min,共30个循环;72℃延伸10min。
反应在Bio-Rad iCycler Thermal Cycler(Bio-Rad Laboratories,Hercules,CA)仪器上进行。扩增产物送交上海生工生物科技有限公司进行测序。测序结果提交到GenBank数据库。
菌株LZU-J-TSL6的16S rRNA基因序列提交到GenBank数据库,利用BLAST搜索同源性较高的序列,用CLUSTAL X 1.81和MEGA 6.0软件进行序列的比对分析并建立系统发育树(图2所示)。可知该菌株隶属于乳杆菌属(Lactobacillus),初步确定该菌为植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6。
2.2菌株初步鉴定与保藏
单克隆菌株LZU-J-TSL6通过革兰氏染色及过氧化氢产气实验初步证明是植物乳杆菌LZU-J-TSL6,该菌株命名为Lactobacillus plantarum LZU-J-TSL6,于2020年10月23日保藏于广东省微生物菌种保藏中心,保藏编号为:GDMCC No:61242,并于2020年10月26日检测为存活状态;保藏地址为:广州市先烈中路100号大院59号楼5楼;联系电话:020-87137633。
实施例3菌株LZU-J-TSL6对模拟胃液和肠液的耐受性
胃液配置:配制pH值为7.4的PBS缓冲液,加入适量的稀盐酸调pH值为3.0,定容至100mL,再称取1.0g胃蛋白酶加入其中,0.22μm滤膜过滤,备用。
肠液配置:配制pH值为8.0的PBS缓冲液,定容至100mL,称取0.3g猪胆盐,1.0g胰蛋白酶,融入其中,0.22μm滤膜过滤,备用。
菌液配置:取甘油保存的菌株LZU-J-TSL6,按2%接种量接种到1.5mL的MRS液体培养基中并置于37℃恒温培养箱中培养10h作为种子菌液用于接下来的实验。种子菌液按2%的接种量接种到5mL的MRS液体培养基中并置于37℃恒温培养箱中培养18h进行二代培养。18h后6000r/min离心10min,弃去上清,沉淀的菌体用同弃去的上清等体积的生理盐水洗涤两次后制成菌悬液,备用。
涂板试验:取1mL的上述菌液接种到9mL的上述已备的胃液中,摇匀置于37℃,90r/min的恒温摇床进行培养。按10倍稀释法进行稀释,并选择合适的稀释度进行涂板,每个稀释度重复两个,在3h各取0.1mL的进行涂板,37℃恒温培养48h后计菌落数;取1mL上述培养3h的胃液加到9mL的已备好的肠液中,摇匀置于37℃,90r/min的恒温摇床进行培养,在3h、6h、9h进行涂板,37℃恒温培养48h后计数。
胃液耐受实验结果如图3所示:与阳性LGG菌相比,菌株LZU-J-TSL6具有一定耐酸耐胆盐的作用,其中,菌株LZU-J-TSL6在pH3.0的胃液中培养三小时之后,其存活率(菌的存活率=接种后某时活菌数/初始的活菌数×100%)在70%以上,其存活率相当于阳性对照组LGG的60倍,说明其具有较高的耐酸性和抗胃蛋白酶的能力,可以在胃液中生长。
肠液耐受实验结果如图4所示:在肠液中培养3h、6h、9h后菌株LZU-J-TSL6的存活率分别为77%、97%、119%表现比较稳定,尤其9h时菌株LZU-J-TSL6在肠液中的存活率(119%)显著高于阳性对照组LGG菌株的存活率(88.8%)。
上述结果表明,本发明提供的菌株LZU-J-TSL6具有较强的胃液和肠液耐受性,可在胃肠液中定植。
实施例4菌株LZU-J-TSL6的抑菌活性
菌株活化:取甘油保存的菌株LZU-J-TSL6,按2%接种量接种到1.5mL的MRS液体培养基中,并置于37℃恒温培养箱中培养10h,作为种子菌液用于接下来的实验。
种子菌液按2%的接种量接种到2mL装有MRS液体培养基的EP管中,并置于37℃恒温培养箱中培养18h进行二代培养后放4℃备用。
指示菌活化:本研究的指示菌株为金黄色葡萄球菌,分别用LB培养基和营养肉汤培养基培养15h,用麦氏比浊法将每个指示菌的浓度调至1-3×108DFU/mL,放4℃冰箱备用。
将已培养18h的LZU-J-TSL6培养液500g离心10min,取上清备用。
取100μL已活化指示菌,调整指示菌的浓度为1.5×108CFU/mL,在相应的培养基上进行涂板,涂布后培养皿置于超净工作台半敞开10min,在固体培养基摆好的牛津杯中加如上述保存的种子菌液上清液200μL,平稳放在37℃培养18h,观察测量每种菌液的抑菌圈直径,平行做三组实验。
抑菌实验如图5所示,菌株LZU-J-TSL6对金黄色葡萄球菌具有一定的抑菌作用,抑菌效果与LGG菌株相当。
综上,本发明提供的植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6具有高产GABA的活性,且具有较强抑菌活性、胃肠液耐受性,可用于制备保健品、食品或食品添加剂。
以上所述仅为本发明的个别示范性实施案例的细节,对于本领域的技术人员来说,本发明在实际应用过程中根据具体的制备条件可以有各种更改和变化,并不用于限制本发明。凡在本发明的精神和原则之内,均应包含在本发明的保护范围之内。
序列表
<110> 兰州大学
<120> 一株高产GABA的植物乳杆菌LZU-J-TSL6及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 952
<212> DNA
<213> 植物乳杆菌(Lactobacillus plantarum)
<400> 1
atggtgtgct atgatgcagt cgacgaactc tggtattgat tggtgcttgc atcatgattt 60
acatttgagt gagtggcgaa ctggtgagta acacgtggga aacctgccca gaagcggggg 120
ataacacctg gaaacagatg ctaataccgc ataacaactt ggaccgcatg gtccgagctt 180
gaaagatggc ttcggctatc acttttggat ggtcccgcgg cgtattagct agatggtggg 240
gtaacggctc accatggcaa tgatacgtag ccgacctgag agggtaatcg gccacattgg 300
gactgagaca cggcccaaac tcctacggga ggcagcagta gggaatcttc cacaatggac 360
gaaagtctga tggagcaacg ccgcgtgagt gaagaagggt ttcggctcgt aaaactctgt 420
tgttaaagaa gaacatatct gagagtaact gttcaggtat tgacggtatt taaccagaaa 480
gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga 540
tttattgggc gtaaagcgag cgcaggcggt tttttaagtc tgatgtgaaa gccttcggct 600
caaccgaaga agtgcatcgg aaactgggaa acttgagtgc agaagaggac agtggaactc 660
catgtgtagc ggtgaaatgc gtagatatat ggaagaacac cagtggcgaa ggcggctgtc 720
tggtctgtaa ctgacgctga ggctcgaaag tatgggtagc aaacaggatt agataccctg 780
gtagtccata ccgtaaacga tgaatgctaa gtgttggagg gtttccgccc ttcagtgctg 840
cagctaacgc attaagcatt ccgcctgggg agtacggccg caaggctgaa actcaaagga 900
attgacgggg gcccgcacaa gcgtgggagc atgtgggtta attcgaagct ac 952
Claims (10)
1.一种植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6,所述植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6于2020年10月23日保藏于广东省微生物菌种保藏中心,保藏编号为:GDMCC No:61242。
2.如权利要求1所述的植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6在生产γ-氨基丁酸中的应用。
3.如权利要求1所述的植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6在制备抑制肠道致病菌药物中的应用。
4.如权利要求3所述的应用,其特征在于,所述肠道致病菌为金黄色葡萄球菌。
5.如权利要求1所述的植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6在制备调节肠道菌群保健品中的应用。
6.一种根据权利要求1所述的植物乳杆菌(Lactobacillus plantarum)LZU-J-TSL6生产γ-氨基丁酸的方法,其特征在于,所述方法为:先将植物乳杆菌(Lactobacillusplantarum)LZU-J-TSL6接入种子培养基中进行种子培养,再接入发酵培养基中进行发酵培养;所述种子培养基为MRS液体培养基,所述发酵培养基为GYP液体培养基。
7.如权利要求6所述的方法,其特征在于,所述种子培养条件为:25-40℃,培养7h以上。
8.如权利要求7所述的方法,其特征在于,所述种子培养条件为:37℃,培养16h。
9.如权利要求6所述的方法,其特征在于,所述发酵培养条件为:将种子培养的产物以高于2%的接种量接入所述发酵培养基中,25-40℃,培养12h以上。
10.如权利要求9所述的方法,其特征在于,所述发酵培养条件为:将种子培养的产物以高于4%的接种量接入所述发酵培养基中,37℃,静置培养48h。
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