CN110144304A - 干酪乳杆菌菌株及其应用 - Google Patents
干酪乳杆菌菌株及其应用 Download PDFInfo
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- CN110144304A CN110144304A CN201910259094.7A CN201910259094A CN110144304A CN 110144304 A CN110144304 A CN 110144304A CN 201910259094 A CN201910259094 A CN 201910259094A CN 110144304 A CN110144304 A CN 110144304A
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- Prior art keywords
- ltl1361
- lactobacillus casei
- bacterial strain
- casei bacterial
- food
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- 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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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
本发明涉及干酪乳杆菌菌株LTL1361及其应用。LTL1361保藏编号是CCTCC M 2019018,可以以活体和/或非活体形式存在,其可以应用于食品、保健品、药品、食品补充剂、化妆品、饲料方面,也可以用于抗菌和消炎,特别是抑制大肠杆菌、金黄葡萄球菌、蜡样芽孢杆菌、福氏志贺氏菌及鼠伤寒沙门氏菌。LTL1361对胃肠道环境的耐受能力强,与肠道上皮细胞的黏附能力强,在人体肠道的定植能力强,安全性高,无脱羧酶活性,对常用的10类抗生素都敏感,具有优良的抑制有害菌的能力。
Description
技术领域
本发明属于生物技术领域;具体涉及干酪乳杆菌菌株及其应用。
背景技术
乳酸杆菌已有很长的食用历史,它维护人体健康和调节免疫功能的作用已被广泛认可,属于公认的安全菌属,在适当条件培养下通常能产生良好的风味,有些菌株甚至能表现出绝佳的产酸能力以及优异的免疫调节功效,大幅提升产品价值。如干酪乳酸杆菌代田株是著名乳酸菌饮料养乐多中采用的一种乳酸杆菌。乳酸杆菌属的特定菌株已被发现能够定植于肠道粘膜,协助并维持人类和动物的健康,例如抗发炎活性和免疫调节活性。
干酪乳杆菌作为益生菌的一种,能够耐受有机体的防御机制,包括口腔中的酶、胃酸和小肠的胆汁酸等。所以干酪乳杆菌进入人体后可以在肠道内大量存活,起到调节肠内菌群平衡、促进人体消化吸收等作用。同时,干酪乳杆菌还具有一定的降血压、降胆固醇的作用,能产生抗体免疫抗体,增强人体免疫力及预防癌症和抑制肿瘤生长等功能。干酪乳杆菌能够抑制和杀死食品中的许多腐败菌及致病菌,并且不影响食物性状,甚至能够改善食品特性。因此筛选性能更佳的干酪乳杆菌对于提升食品品质,提高食品价值有重要的意义。
发明内容
本发明的第一个目的在于提供一株干酪乳杆菌菌株。
本发明的第二个目的是提供以上干酪乳杆菌菌株的应用。
实现上述第一个发明目的的技术方案是:所述干酪乳杆菌菌株的保藏编号是CCTCC M 2019018,其核苷酸包含SEQ ID NO:1至SEQ ID NO:51所示的特征序列。
该干酪乳杆菌菌株可以以活体形式存在,也可以非活体形式存在。活体形式存在优选以冻干粉形式存在。
实现上述第二个发明目的的技术方案是:所述干酪乳杆菌菌株在食品、保健品、药品、食品补充剂、化妆品、饲料方面的应用;也可以用于抗菌和消炎,特别是抑制大肠杆菌、金黄葡萄球菌、蜡样芽孢杆菌、福氏志贺氏菌及鼠伤寒沙门氏菌。
本发明的干酪乳杆菌菌株LTL1361对胃肠道环境的耐受能力强,经过模拟胃肠道环境作用4h后,菌株存活率仍然很高;与肠道上皮细胞的黏附能力强,其在人结直肠癌细胞—— Caco-2细胞上的粘附数比现有干酪乳杆菌明显要多,说明LTL1361菌株在人体肠道的定植能力强;而且LTL1361安全性高,无脱羧酶活性,对常用的10类抗生素都敏感,具有优良的抑制有害菌的能力。
附图说明
图1粪便样品稀释液在改良的MRS固体培养基上培养后的典型菌落
图2有溶钙圈或浅蓝色的单菌落划线多代纯化结果
图3 LTL1361特异性引物PCR验证结果
图4 LTL1361核苷酸PCR扩增产物琼脂糖凝胶电泳结果
图5 LTL1361全基因组图谱
图6 LTL1361菌株生长曲线
图7 LTL1361体外粘附人结直肠癌细胞Caco-2图
图8 LTL1361体外粘附人结直肠癌细胞Caco-2实验结果
图9 LTL1361氨基酸脱羧酶活性检测,其中A为精氨酸,B为赖氨酸,C为酪氨酸,D为组氨酸
生物材料保藏信息
LTL1361,分类命名为干酪乳杆菌(Lactobacillus casei),已于2019年1月4日在位于湖北省武汉市武昌区八一路珞珈山的中国典型培养物保藏中心保藏,保藏编号为CCTCC M 2019018。
具体实施方式
本发明具体实施方式中的试验结果数据皆以平均值±标准差(Means±SD)表示。统计分析使用单因子变异数分析[One-way analysis of variance(One-way ANOVA)]。“+”表示90%菌株阳性;“-”表示90%菌株阴性;“d”表示11%~89%菌株阳性。
本发明具体实施方式中,“NCBI”表示美国国家生物技术信息中心。“BLAST”是“Basic Local Alignment Search Tool”的缩写,一种生物信息分析软件名称。所有核苷酸的测序单位是生工生物工程(上海)股份有限公司,使用的测序仪器是Illumina公司的Hiseq-2500 150PE测序平台。
本发明实施例中所采用的实验方法,如果没有给出特殊说明,均为本领域常规方法;所用的实验材料,如果没有给出特殊说明,说明可从商业途径获得。
实施例1菌种筛选与鉴定
1.菌种筛选与鉴定
收集广西巴马县2个月以内未服用任何抗生素或乳酸杆菌制剂,且居住地位置分散、年龄在100岁~128岁之间的健康老人粪便样品,冷冻保藏。粪便样品解冻后用无菌磷酸盐缓冲溶液溶解并稀释成梯度浓度,涂布在改良的MRS琼脂平板上,37℃培养。培养结果见图1。所述“改良的MRS固体培养基”是指MRS固体培养基中含碳酸钙(CaCO3)和5-溴-4-氯-3-吲哚-β -D-半乳糖苷(X-gal),CaCO3的质量百分比浓度是0.8%,X-gal的浓度是每200mL培养基含2%X-gal 200μl。从图1可以观察到形成的菌落形态为圆形、乳白色、在改良的MRS琼脂平板上形成钙溶圈、不透明、表面光滑、边缘圆整、凸起、直径小于1.5mm。挑取有溶钙圈或浅蓝色的单菌落划线多代纯化。纯化结果见图2。纯化得到的菌株编号为LTL1361,用40%甘油于-20℃和-80℃下分别保存,备用。革兰氏染色后观察到细胞形态为杆菌、革兰氏阳性菌,或丛生、或对生。根据《伯杰士细菌学手册》乳杆菌属部分菌落形态及细胞形态特征,LTL1361符合乳杆菌形态学鉴定特征,为疑似乳酸杆菌。
对LTL1361进行过氧化氢酶试验、葡萄糖产酸产气试验、明胶液化试验、硝酸盐还原试验、石蕊牛乳试验、温度耐受试验、pH试验(结果见表1),以及糖类发酵试验(结果见表2)。根据《乳酸细菌分类鉴定及实验方法》,由表1、表2的实验结果可初步鉴定LTL1361 为干酪乳杆菌(Lactobacillus casei)。
表1 LTL1361生理生化实验鉴定结果
表2 LTL1361糖发酵实验结果
2.特异性引物PCR验证LTL1361
为了鉴定LTL1361,进一步对LTL1361进行特异性引物聚合酶链反应(PolymeraseChain Reaction,PCR)验证。
设计的特异性引物如下:
F:5’-AGC AGT AGG GAA TCT TCC A-3’
R:5’-ATT TCA CCG CTA CAC ATG-3’
PCR扩增后,PCR产物琼脂糖凝胶电泳,Marker采用100bp DNA marker。电泳结果见图3。干酪乳杆菌特异性引物PCR后,片段长度为344bp,图3在相应处出现了荧光条带,且无明显拖尾现象,说明PCR成功,从特定引物角度确认了上述生理生化和糖发酵实验鉴定结果。
3. 16S rDNA序列鉴定
再对LTL1361的16S rDNA核甘酸序列进行鉴定。提取LTL1361基因组DNA,将提取的LTL1361基因组DNA作为模板进行聚合酶链反应(Polymerase Chain Reaction,PCR)。
通用引物如下:
27F:5′-AGAGTTTGATCCTGGCTCAG-3′
1492R:5′-GGCTACCTTGTTACGACTT-3′
扩增产物的琼脂糖凝胶电泳结果见图4。图4显示在约1500bp处出现了荧光条带,无明显拖尾现象,说明PCR扩增成功,满足16S rDNA序列测定需要。序列测定结果与美国国家生物技术信息中心(National Center for Biotechnology Information,简称NCBI)数据库进行 BLAST比对,结果表明LTL1361与干酪乳杆菌标准菌株ATCC334相似度达100%。
综合LTL1361形态特征、生理生化实验、糖发酵实验、特异性引物PCR验证结果以及16S rDNA序列分析结果,将LTL1361确定为干酪乳杆菌(Lactobacillus casei)。
实施例2 LTL1361的特征序列
LTL1361 DNA序列检测结果显示LTL1361基因组由1条染色体和2个质粒组成,其中染色体基因组为环状,平均鸟嘌呤(G)和胸嘧啶(C)之和含量为46.22%,长度为3,110,507bp,含3125个独立编码区(CDSs),平均长度为854bp,编码区总长度占全基因组的比例为85.8%。LTL1361全基因图见图5。
在所有3125个独立编码基因中,有1911个基因能够归类至直系同源群(Clusterof Orthologous Group,COG)功能分类数据库中,参与糖转运和代谢相关的基因有283个,参与复制、重组和修复的基因有94个,参与氨基酸转运和代谢的基因有187个,参与功能代谢的基因有93个,参与无机盐转运和代谢的基因有108个,另外130个基因功能未知,被定义为假定蛋白,994个基因与COG数据库中的基因无任何匹配。
将LTL1361核苷酸序列与NCBI数据库比对,LTL1361基因组DNA中有51段核苷酸序列体现了LTL1361基因组的专有特征,这51段特征序列如SEQ ID NO:1~SEQ ID NO:51所示。
实施例3 LTL1361生长曲线和传代周期
LTL1361的生长曲线在MRS液体培养基中测定,测定结果如图6。图6表明LTL1361生长平缓,1%接种量下,0-3h为生长延滞期,3-14h为生长对数期,14h后进入基本稳定期。
实施例4 LTL1361胃肠道耐受能力
将LTL1361重悬于pH 2.5人工胃液(NaCl 0.2g/100mL、胃蛋白酶0.32g/100mL,再用浓度为1mol/L HCl调整pH值为2.5)中,37℃震荡培养120min,离心,收集菌体,再将菌体重悬于pH值为8.0人工肠液(KH2PO4 0.68g/100mL、胰蛋白酶1g/100mL、胆盐 0.3g/100mL,pH值为8.0)中,37℃摇床震荡培养120min。检测处理前后样品中的活菌数目。活菌数表达单位是CFU/mL。
乳酸菌发挥生理功能特性的活菌数临界值为106CFU/mL。将本发明菌株初始菌数调整到 108CFU/mL,模拟胃肠环境培养4h,菌株活菌数仍然在108CFU/mL以上(见表3),说明本发明菌株消化道逆环境耐受性良好,能经受胃肠道逆环境在人体内发挥益生作用,可以作为潜在的益生菌使用。
表3 LTL1361对模拟消化道240min耐受性结果
实施例5 LTL1361对Caco-2细胞黏附能力
Caco-2人结直肠癌细胞由中国科学院上海生命科学研究院细胞资源中心提供;DMEM高糖培养基(含L-谷氨酰胺)、青链霉素双抗:HyClone产品;胎牛血清:BiologicalIndustries 产品;胰蛋白酶-EDTA:Neuronbc产品;细胞复苏培养液:80%DMEM(含L-谷氨酰胺)、 20%胎牛血清、青链霉素双抗100IU/mL;细胞生长维持液:90%DMEM(含L-谷氨酰胺)、10%胎牛血清,加1%(v/v)青链霉素双抗或不加青链霉素双抗。
将传代至性状稳定的Caco-2细胞用胰蛋白酶-EDTA消化后,再用细胞生长维持液调整细胞悬浮液,然后加入已置有无菌盖玻片的6孔板中,每两天观察一次并换液,镜检至细胞铺满整个孔板底部,形成均匀的单层细胞后,吸尽上层细胞废液,用无菌磷酸缓冲液溶液吹洗孔板底部,减少青链霉素双抗对菌体的影响。每孔加入LTL1361(108CFU/mL)-生长维持液 (不含青链霉素双抗),37℃、5%CO2-95%空气恒温二氧化碳培养箱中孵育,倾斜孔板,吸尽上液,用巴氏吸管吸取无菌磷酸盐缓冲液冲洗盖玻片,除去未结合的LTL1361,晾干后用多聚甲醛固定,革兰氏染色。染色后的盖玻片于100倍油镜下观察,随机选取25个视野,每个视野选取4个挑选细胞面积接近的Caco-2细胞团计数取平均值。结果见图7和图8。由图7和图8的结果可知,LTL1361的粘附能力非常高,平均粘附数为24 CFU/细胞,说明 LTL1361在肠道内的定植能力强。
实施例6 LTL1361抗生素药敏实验表型测试
将液态扩培的LTL1361按美国临床实验室标准化委员会(NCCLS)要求用无菌生理盐水重悬至浓度调整为1×108~2×108CFU/mL,然后将LTL1361菌液涂布于改良药敏实验琼脂上(90%标准药敏检测肉汤、10%MRS),晾干表面水分后再将抗生素纸片——阿莫西林(10 μg/片)、链霉素(10μg/片)、氨苄西林(10μg/片)、利福平(5μg/片)、林可霉素(2μg/片)、罗红霉素(15μg/片)、庆大霉素(10μg/片)、四环素(30μg/片)、青霉素(10μg/ 片)、氯霉素(30μg/片)、红霉素(15μg/片)、阿奇霉素(15μg/片),按NCCLT-LS操作标准贴于实验琼脂上,平行5次,37℃下培育48h后测量抑菌直径大小(单位是mm)。参照NCCLS条款判断药敏结果,结果见表4。表4表明LTL1361仅对链霉素和氨苄西林表现为适中,对其余10种常用抗生素表现为敏感。
表4 LTL1361抗生素药敏感性表型实验结果
实施例7氨基酸脱羧酶活性检测
根据《微生物培养基的制造与应用》制备改良氨基酸脱羧酶活力检测固体培养基。检测的氨基酸为:精氨酸、酪氨酸、赖氨酸和组氨酸。阳性对照菌为大肠杆菌ATCC2522。将LTL1361以及阳性对照菌ATCC2522的单菌落用接种环涂至对应培养基上,37℃培养48h,观察颜色变化。如果为紫色,则为阳性结果,说明该菌株具备氨基酸脱羧酶活性,能产生有害胺类物质;如果为黄色,则为阴性结果,说明说明该菌株不具备氨基酸脱羧酶活性,不能产生有害生物胺。LTL1361氨基酸脱羧酶活性检测结果见图9。由图9可知,LTL1361氨基酸脱羧酶活性检测结果为阴性,表明LTL1361不具备产生对应氨基酸脱羧酶和对应有害生物胺的能力。
实施例8 LTL1361抑菌能力
以大肠杆菌ATCC2522、金黄葡萄球菌、蜡样芽孢杆菌、福氏志贺氏菌及鼠伤寒沙门氏菌为实验菌,这5种菌来源于本实验室或中国医学细菌保藏管理中心。
分别将大肠杆菌ATCC2522、金黄葡萄球菌、蜡样芽孢杆菌、福氏志贺氏菌及鼠伤寒沙门氏菌接种至液态LT-LB肉汤培养基中,37℃过夜培养,传3-5代至性状稳定。将LTL1361接种至MRS液态培养基,培养至平台期后,离心取上清液。将液态扩培后至相同浓度的5种致病菌按0.1%(v/v)接种至35mL LB肉汤琼脂培养基中,混匀后倾倒至培养皿中。凝固后打孔,每个培养皿等距离打四个孔。每个孔中注入已灭菌的1.5%热琼脂20μL,凝固后使用。其中1个孔注入空白培养基,另外3个孔注入LTL1361培养液上清液,每孔每次注入50μL对应液体,超净台内晾干,重复注入3次,每孔总体积为150μL。每种致病菌平行3次。4℃培养2h后37℃培养36h,然后测量抑菌圈大小。测量结果见表5。表5说明LTL1361对五种致病菌具有明显抑制能力。
表5株菌培养液上清液对5种致病菌的抑菌圈大小
实施例9 LTL1361在食品、保健品、药品、食品补充剂、药品方面的应用
本发明LTL1361在食品、保健品、药品、食品补充剂、药品、化妆品、饲料方面有着广泛的应用。
作为优选之一是利用干酪乳杆菌LTL1361制备乳酸菌饮料。将脱脂乳粉用蒸馏水配制成 12%的复原脱脂乳,95℃下杀菌120min,冷却至37℃后,在无菌条件下,按3%的接种量接种干酪乳杆菌LTL1361,37℃恒温发酵72h,终点酸度控制在165~175°T。将15%白砂糖溶于适量水中,95℃杀菌5min,冷却至20℃,制得糖液。发酵完成后将发酵液与糖液以1∶4的比例混合均匀,并于20~25MPa下进行均质,然后72℃热处理15s,冷却至15℃时进行灌装,无菌灌装后于4℃贮藏,即得乳酸菌饮料。
作为优选之二是利用干酪乳杆菌LTL1361生产发酵大豆乳。挑选表面光滑无霉变的大豆,用25℃蒸馏水浸泡12h,至大豆的含水量至60%左右,大豆与蒸馏水之比为1∶8,然后用0.5%的NaHCO3溶液浸泡2h,清洗去皮,热水磨浆去渣,将豆浆在100℃下煮沸10min,添加6%的蔗糖,调pH至7.0,均质10min,121℃杀菌15min,待温度冷却至37℃,按5%的接种量接种干酪乳杆菌LTL1361,37℃恒温发酵,把发酵终止后的大豆酸乳放入4℃冰箱, 12h后熟,制成干酪乳杆菌发酵大豆乳产品。
作为优选之三是利用干酪乳杆菌LTL1361生产益生菌酸奶。新鲜牛乳中加入8%的蔗糖,充分搅拌均匀,完全溶解后在20~25MPa压力下均质5min,然后95℃杀菌5min,待温度冷却至37℃,按2%的接种量接种干酪乳杆菌LTL1361,37℃恒温发酵6h,发酵结束的益生菌酸奶置于4℃冰箱,后熟12h进行无菌灌装,即得益生菌酸奶成品。
以上所述仅是本发明的优选实施方式,本技术领域普通技术人员在不脱离本发明原理的前提下,还可以做出若干改进,这些改进也应视为本发明的保护范围。
Claims (8)
1.一株干酪乳杆菌菌株,其特征是:保藏编号是CCTCC M 2019018。
2.权利要求1所述干酪乳杆菌菌株,其特征是:包含SEQ ID NO:1至SEQ ID NO:51所示的特征序列。
3.权利要求1所述干酪乳杆菌菌株,其特征是:以活体形式存在。
4.权利要求3所述干酪乳杆菌菌株,其特征是:以冻干粉形式存在。
5.权利要求1所述干酪乳杆菌菌株,其特征是:以非活体形式存在。
6.权利要求1-5任一项所述干酪乳杆菌菌株,其特征是:应用于食品、保健品、食品补充剂、药品、化妆品、饲料。
7.权利要求6所述干酪乳杆菌菌株,其特征是:应用于抗菌和消炎。
8.权利要求6所述干酪乳杆菌菌株,其特征是:用于抑制大肠杆菌、金黄葡萄球菌、蜡样芽孢杆菌、福氏志贺氏菌及鼠伤寒沙门氏菌。
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