CN110452830B - 发酵乳杆菌菌株及其应用 - Google Patents
发酵乳杆菌菌株及其应用 Download PDFInfo
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Abstract
本发明涉及发酵乳杆菌菌株LTP1332及其应用。该菌株保藏编号是CCTCCM 2019029,可以以活体形式存在,也可以以非活体形式存在,活体形式存在优选以冻干粉形式存在,可以应用在食品、保健品、药品、食品补充剂、药品方面的应用,优选用于消炎抗菌和益生菌。LTP1332对胃肠道环境的耐受能力强,具有较强的胃肠道耐受能力,能够顺利通过机体胃肠道,发挥益生功能,具有较好的修复结肠黏膜损伤能力,作为益生菌能够在DSS诱导的小鼠体内发挥抗炎作用,下调促炎症细胞因子的水平,且效果优于模式菌株CCIC22537。
Description
技术领域
本发明属于生物技术领域;具体涉及发酵乳杆菌菌株及其应用。
背景技术
发酵乳杆菌(Lactobacillus fermentum)属于乳杆菌科中的乳杆菌属,是人和动物肠道、 口腔和阴道的正常菌群,具有维持肠道内微生物群稳定、改善宿主机体内环境的能力,是促 进人体健康最主要的乳酸杆菌种类之一。
发酵乳杆菌发挥抑菌作用的途径主要有:发酵产生的有机酸降低机体微环境pH值,抑 制不耐酸的致病菌和腐败菌;与致病菌竞争胃肠道结合位点和营养物质,防止致病菌的粘附定 植。另外,发酵乳杆菌还具有抗氧化活性、免疫活性、降胆固醇、降血压、调节肠道菌群结 构等多种益生功能。研究表明,益生菌定植肠道后能修复粘膜损伤,刺激隐窝细胞增殖,调 节炎症因子的表达水平。发酵乳杆菌作为形成健康肠道菌群的重要益生菌之一,也被作为治 疗炎症性肠病的候选菌。此外,发酵乳杆菌还被用于治疗过敏性皮肤炎、抗生素相关性腹泻 等多种疾病。发酵乳杆菌在食品领域,多用于豆乳和香肠等发酵食品中。用发酵乳杆菌发酵 豆乳时,不仅能够赋予产品以独特的风味、保留大豆本身的营养成分,还能使产品具有降胆 固醇、调整肠道菌群等益生功能。另外,发酵乳杆菌所具有的抑菌性能够抑制食品中腐败菌 的生长,延长产品的货架期。
虽然发酵乳杆菌及其产品的应用已经涉及到医疗保健、食品工业、畜牧生产等多个领 域,但是真正高质量、符合市场需求的发酵乳杆菌产品相对较少。
发明内容
本发明的第一个目的在于提供一株发酵乳杆菌菌株。
本发明的第二个目的是提供以上发酵乳杆菌菌株的应用。
本发明的第三个目的是提供一种发酵乳杆菌培养基。
所述发酵乳杆菌菌株的保藏编号是CCTCC M 2019029,其核苷酸包含SEQ ID NO:1至 SEQ ID NO:16所示的特征序列。
该发酵乳杆菌菌株可以以活体形式存在,也可以以非活体形式存在。活体形式存在优选 以冻干粉形式存在。
所述发酵乳杆菌菌株在食品、保健品、药品、食品补充剂、药品方面的应用,所述应用 优选用于消炎抗菌和益生菌。
本发明发酵乳杆菌菌株LTP1332对胃肠道环境的耐受能力强,在4h模拟胃液中的存活率 为72.03%,之后在12h模拟肠液中的存活率为47.63%,远远超过了发挥益生功能的活菌数要 求,说明LTP1332具有较强的胃肠道耐受能力,能够顺利通过机体胃肠道,发挥益生功能。 LTP1332具有较好的修复结肠黏膜损伤能力,作为益生菌能够在DSS诱导的小鼠体内发挥抗 炎作用,下调促炎症细胞因子的水平,且效果优于模式菌株CCIC22537。
附图说明
图1 LTP1332的细胞形态
图2 LTP1332特异性引物PCR验证结果
图3 LTP1332核苷酸PCR扩增产物琼脂糖凝胶电泳结果
图4 LTP1332全基因组图谱
图5 LTP1332菌株生长曲线
图6 LTP1332制剂在模拟胃液中耐受性
图7 LTP1332制剂在模拟肠液中耐受性
图8各组小鼠每日体重变化,*与模型组相比,具有显著差异(P<0.05),**与模型组相 比,具有极显著差异(P<0.01);#与健康对照组相比,具有显著差异(P<0.05),##与健康 对照组相比,具有极显著差异(P<0.01)。
图9结肠病理组织切片,A:DSS+CCIC22537组,B:DSS+LTP1332组,C:DSS组; D:DSS+SASP组;E:健康对照组;ME:外肌层,SM:粘膜下层,MM:正常粘膜,Cr: 隐窝,GC:杯状细胞,○:炎症因子浸润。
图10组织学损伤程度评分
图11结肠细胞中促炎症因子TNF-α的含量(n=10)
图12结肠细胞中促炎症因子IFN-γ的含量(n=10)
LTP1332,分类命名为发酵乳杆菌LTP1332(Lactobacillus fermentiumLTP1332),已于 2019年1月8日在位于中国.武汉.武汉大学的中国典型培养物保藏中心保藏,保藏编号为 CCTCC NO:M 2019029。
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。*与模型组相比,具有显著 差异(P<0.05),**与模型组相比,具有极显著差异(P<0.01);#与健康对照组相比,具有 显著差异(P<0.05),##与健康对照组相比,具有极显著差异(P<0.01)。
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,以2%接种量接种到改良的MRS固体培养基上,37℃培养。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。
挑取有溶钙圈或浅蓝色的单菌落划线多代纯化。纯化得到的菌株编号为LTP1332。革兰 氏染色后观察到细胞为革兰氏阳性菌,棒状,直径0.5-0.9μm,大多数单个生长,也有成对出 现。见图1。
对LTP1332进行过氧化氢酶实验、明胶液化实验、硝酸盐还原试验、石蕊牛奶实验、葡 萄糖产酸产气实验、碳水化合物发酵产酸试验(结果见表1和表2)并检测LTP1332接种于pH 4.5、pH 7.0和pH 9.0的改良MRS液体培养基,和在15℃、45℃下的改良MRS液体培养 基培养的生长情况。参照《伯杰士细菌学手册》及《乳酸细菌分类鉴定及实验方法》中乳杆 菌属部分,对LTP1332初步鉴定,结果为发酵乳杆菌(Lactobacillus fermentum)。
表1 LTP1332菌株的生理生化实验鉴定结果
表2 LTP1332菌株的糖发酵实验结果
特异性引物PCR验证LTP1332
进一步对LTP1332进行特异性引物聚合酶链反应(Polymerase Chain Reaction,PCR)验 证。扩增产物的琼脂糖凝胶电泳结果见图2。发酵乳杆菌专用引物特异性PCR后片段长度分 别为337bp,图2中相应处出现荧光条带,且无明显拖尾现象,说明PCR扩增成功,并从特 定引物角度确认了上述生理生化和糖发酵实验鉴定结果。
设计的特异性引物如下:
F:5’-AAT ACC GCA TTA CAA CTT TG-3’
R:5’-GGT TAA ATA CCG TCA ACG TA-3’
2. 16S rDNA序列鉴定
利用通用引物对LTP1332的16S rDNA PCR扩增,相应基因序列扩增产物电泳结果见图 3。图3显示在约1500bp处出现荧光条带,且无明显拖尾现象,说明PCR扩增成功,能满足后 续16S rDNA测序需要。
通用引物如下:
27F:5′-AGAGTTTGATCCTGGCTCAG-3′
1492R:5′-GGCTACCTTGTTACGACTT-3′
将LTP 1332的16S rDNA序列与GenBank数据库中已知乳酸杆菌标准菌株的16SrDNA序 列进行同源性比对(结果见表3)。表3表明LTP1332与干酪乳杆菌IFO 3956的同源百分比高 于99%。
表3 LTP1332与GenBank数据库中标准菌株的基因序列同源性比对结果
实施例2 LTP1332的特征序列
LTP1332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,鸟嘌呤(G) 和胸嘧啶(C)之和的平均含量为51.3%,长度为2,149,768bp,含2,218个独立编码区(CDSs),平均长度为835bp,编码区总长度占全基因组的比例为86.18%。LTP1332全基因 图见图4。
将LTP1332核苷酸序列与NCBI数据库比对,LTP1332基因组DNA中有16段核苷酸序列,体现了LTP1332基因组的专有特征,这16段特征序列如SEQ ID NO:1~SEQ ID NO:16 所示。
实施例3 LTP1332生长曲线和传代周期
以1%接种量将LTP1332种子液接种于MRS液体培养基,每隔3h测定发酵液中活菌数 及还原糖含量,以发酵时间(h)为横坐标,以活菌数对数值(Log CFU/mL)和还原糖含量(g/L)为纵坐标,绘制如图5所示的发酵乳杆菌LTP1332生长曲线。由图5得出,培养6 h,LTP1332进入生长对数期,24h左右进入稳定期,菌体浓度达到最大,24~39h,菌体浓 度无明显变化,39h菌体浓度开始下降,进入衰亡期。
实施例4 LTP1332在模拟胃液中耐受性
取LTP1332冻干菌粉,溶解于pH 2.5的人工模拟胃液中,37℃恒温培养4h,测定活菌 数。收集模拟胃液中的菌体,重悬于人工模拟肠液,37℃恒温培养,测定活菌数。活菌数表 达单位是CFU/mL。结果见图6和图7。人工模拟胃液的组成为NaCl 2.0g/L、胃蛋白酶(pepsin 1∶3000)3.2g/L,调整pH值为2.5,过滤除菌。人工模拟肠液组成为KH2PO4 6.8g/L、胰蛋白 酶(Trypsin 1∶250)1.0g/L、胆盐0.3g/L,调整pH值为8.0。
图6和图7表明经过4h模拟胃液处理后,LTP1332存活率为72.03%,再经12h模拟肠液处 理后最终的存活率为47.63%,说明LTP1332具有较强的胃肠道耐受能力,能够顺利通过胃肠 道,在体内发挥其益生作用。
实施例5 LTP1332在DSS诱导小鼠结肠炎模型中的效果
将LTP1332和模式菌株CCIC 22537(购自中国工业微生物菌种保藏中心)分别于MRS 培养基37℃恒温培养,离心收集菌体,重悬于无菌生理盐水中,利用分光光度计配制菌体浓 度约为2×109CFU/mL的菌悬液。
将50只C57BL/6J小鼠随机分为5组,分别是正常对照组、右旋葡聚糖硫酸钠(DSS)模型组、CCIC22537组、LTP1332组、柳氮磺胺吡啶(SASP)组,每组10只,雌雄各半。 正常对照组用无菌生理盐水灌胃;DSS模型组用DSS造模;CCIC22537组(模式菌株对照 组)用DSS造模加灌胃2×109CFU/mL CCIC22537;LTP1332组用DSS造模加灌胃2×109 CFU/mL LTP1332;SASP组(阳性对照组)用DSS造模加灌胃200mg/(kg·d)SASP。DSS造 模方法:3.0%DSS溶液(w/v)自由饮用7天。生理盐水、试验菌株LTP1332以及SASP的 灌胃从造模开始前两天开始进行。
每天观察小鼠进食、活动等情况,记录体重变化,并观察粪便性状及是否隐血。图8为 造模后各组小鼠体重变化。造模第三天,除正常组小鼠体重略有上升外,其余各组开始迅速 下降,DSS模型组的下降幅度最大,其次是CCIC22537组;造模第五天LTP1332组、SASP组、CCIC22537组小鼠体重下降速度均减缓,试验结束时,模型组小鼠体重下降了 3.2±1.1g,与正常组小鼠体重的改变相比有显著差异(P<0.05),说明用DSS诱导小鼠结肠 炎有效。LTP1332组和SASP组小鼠体重改变与DSS模型组相比,有显著性差异 (P<0.05),CCIC22537组小鼠体重改变比模型组幅度减小,但差异无统计学意义,说明 LTP1332能够缓解由DSS引起的小鼠体重下降,且效果优于模式菌株CCIC22537。
按照Bauer的DAI评分标准(表4)计算DAI积分,初步评估造模情况及结肠炎的严重程度。其中,DAI=便血分数+大便性状分数+体重下降分数。脾是参与免疫反应的重要淋巴器 官。DSS诱导的小鼠结肠炎能够增加小鼠脾的活动,从而引起脾重量增加。试验结束后测得 的各组小鼠DAI积分及脾脏系数结果如表5所示。其中脾脏系数=脾脏重量(mg)/体重(g)。
表4疾病活动指数(DAI)评分标准
表5各组小鼠DAI评分,脾脏系数的比较(x±s,n=8)
组别 | DAI | 脾脏系数/% |
DSS+CCIC 22537 | 6.75±0.69<sup>*##</sup> | 3.83±0.25<sup>##</sup> |
DSS+LTP1332 | 4.53±1.06<sup>**##</sup> | 3.93±0.26<sup>##</sup> |
DSS | 7.95±1.37<sup>##</sup> | 4.20±0.32<sup>##</sup> |
DSS+SASP | 3.43±1.24<sup>**##</sup> | 3.40±0.33<sup>##</sup> |
正常对照组 | 0 | 2.14±0.18 |
DSS模型组小鼠DAI积分比正常组显著升高(P<0.01),CCIC22537、LTP1332、SASP的干预均能降低小鼠DAI积分,SASP的干预效果最好,LTP1332其次,CCIC22537效果略 差,但三组小鼠DAI积分较模型组均有显著差异(P<0.01)。DSS、CCIC22537、LTP1332、 SASP四组小鼠的脾脏系数较正常对照组显著升高,说明造模引起小鼠脾脏肿胀。
试验结束后,取出小鼠肛门至盲肠末端整个结肠和直肠段,用预冷的生理盐水清洗结 肠。取结肠1cm(距肛门1cm处开始),用4%多聚甲醛固定,石蜡包埋、切片、HE染色, 镜检观察。图9是小鼠结肠病理学切片图。正常组小鼠肠黏膜上皮完整,腺体结构排列规 则,隐窝正常,无充血、水肿、糜烂、溃疡(图E)。DSS模型组小鼠隐窝破坏变形,腺体 紊乱,杯状细胞减少,大量中性粒和淋巴细胞浸润,主要累及粘膜、粘膜下层,少数可达浆 膜层(图C)。SASP(图D)、LTP1332(图B)、CCIC22537(图A)三组小鼠的结肠粘 膜病理学损伤均有不同程度的缓解,结肠粘膜缺失较少、组织结构较完整,隐窝炎和隐窝脓 肿明显减少,病变范围缩小,炎症细胞浸润亦显著减少。
参考Dieleman等的评分标准,用以下四个方面的乘积表征组织学损伤程度,包括:炎症 (无记0分;轻度记1分;重度记2分)、病变深度(无记0分;粘膜下层记1分;肌层记2分;浆膜层记3分)、隐窝破坏评分(无记0分;基底1/3隐窝破坏记1分;基底2/3隐窝破坏记2分;仅有完整表面上皮记3分;全部隐窝和上皮被破坏记4分)、病变范围(1-25%记1分;26-50%记2分;51-75%记3分;76-100%记4分)。结果见图10。图10中DSS模型组小鼠的组织学损伤评分显著高于正常组(P<0.01),CCIC22537组、LTP1332组、SASP组评分均显著降低 (P<0.01),各组排序为:SASP<LTP1332<CCIC22537。说明LTP1332具有较好的修复结 肠黏膜损伤能力。
取小鼠结肠组织,剥除浆膜层和肌层,将分离好的新鲜结肠黏膜固定于UssingChamber 装置上,整个装置处于37℃恒温环境,并持续向两侧液体中通入95%氧气和5%二氧化碳混合 气体直至试验结束,两侧分别加入5mL的Kreb液(2.25mmol/L KH2PO4,108mmol/L NaCl, 3mmol/L KCl,2mmol/L CaCl2·2H2O,22mmol/L NaHCO3,8.9mmol/L葡萄糖,pH7.4)。 调节电压钳零电压,以1mV刺激电压每隔60s刺激0.5s,待系统稳定后,记录跨膜电阻抗 (TER)。结果见表6
表6各组小鼠结肠上皮跨膜电阻值(n=5)
DSS模型组小鼠结肠上皮TER值明显低于正常组(P<0.05),结合图9(E)DSS组小鼠结肠切片可以判断,DSS组小鼠的肠上皮粘膜屏障受到了损伤,上皮细胞旁通透性增加。CCIC22537、LTP1332、SASP组小鼠的结肠上皮TER值较DSS组均有不同程度的增加 (P<0.05),其中LTP1332干预组增加幅度明显高于SASP组和CCIC22537干预组,说明该菌 株具有较强的修复肠粘膜损伤能力。
采用ELISA法测定各组小鼠结肠样品中促炎症细胞因子IFN-γ、TNF-α的浓度。TNF-α浓 度的检测结果见图11,IFN-γ浓度的检测结果见图12。图11和12中DSS模型组小鼠的IFN-γ、 TNF-α水平较正常组显著升高(P<0.01)。SASP组、LTP1332组的IFN-γ、TNF-α浓度较模型 组均显著降低(P<0.05),CCIC22537组的TNF-α浓度较模型组差异显著(P<0.05),而IFN- γ浓度差异不显著。可知,DSS诱导的急性溃疡性结肠炎模型会增加小鼠体内炎症细胞因子的 水平,LTP1332、CCIC22537、SASP的干预均能不同程度地降低IFN-γ、TNF-α浓度。 LTP1332、CCIC22537作为益生菌能够在DSS诱导的小鼠体内发挥抗炎作用,下调促炎症细胞 因子的水平,且LTP1332效果优于CCIC22537。
实施例6冻干菌粉制备
将LTP1332发酵菌液6000rpm离心10min去上清,经无菌生理盐水洗涤两次后,获得菌 泥,用原发酵液1/10体积的生理盐水重悬,将保护剂和菌泥分别按照8∶1比例混合,放入真空 冷冻干燥机中冷冻干燥24h,得LTP1332冻干菌粉。复水后采用平板计数法检测冻干存活率及 每克冻干菌粉中的活菌数。其中保护剂为脱脂乳、海藻糖、L-谷氨酸钠、吐温80的混和物, 其比例为脱脂乳120g/L,海藻糖70g/L,L-谷氨酸钠10g/L,吐温8010g/L。此条件下得到的 冻干菌存活率为75.21±1.42%,远高于未添加保护剂的菌体冻干后存活率。
Claims (3)
1.一株发酵乳杆菌(Lactobacillus fermentium)菌株,其特征是:保藏编号是CCTCCNO:M2019029。
2.权利要求1所述发酵乳杆菌(Lactobacillus fermentium)菌株在制备抗结肠炎的药物中的应用。
3.权利要求1所述发酵乳杆菌(Lactobacillus Fermentium)菌株在制备食品中的应用。
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