CN114657105B - 一株产萝卜硫素且能够缓解炎症的长双歧杆菌ccfm1206 - Google Patents
一株产萝卜硫素且能够缓解炎症的长双歧杆菌ccfm1206 Download PDFInfo
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Abstract
本发明公开了一株产萝卜硫素且能够缓解炎症的长双歧杆菌CCFM1206,属于微生物技术领域。本发明提供的长双歧杆菌CCFM 1206可将转化萝卜硫苷转化为萝卜硫素,促进哺乳动物体内萝卜硫苷的代谢和萝卜硫素的生成。本发明的长双歧杆菌CCFM1206可单独或与含萝卜硫苷联合用于缓解溃疡性结肠炎期间体重的减轻,降低结肠促炎因子的释放,改善结肠屏障功能,缓解全身性炎症引起的脾脏肿大,降低血清中促炎因子的水平,减少肝脏组织中促炎因子的含量和提高抗炎因子的含量,提高短链脂肪酸的含量。
Description
技术领域
本发明涉及一株产萝卜硫素且能够缓解炎症的长双歧杆菌CCFM1206,属于微生物技术领域。
背景技术
萝卜硫素,分子式为C6H11NOS2,是一种异硫氰酸酯,是十字花科植物中的硫代葡萄糖苷(主要是萝卜硫苷)的次级代谢物。然而,萝卜硫素通常并不存在于天然植物中,而是以它的前体物质萝卜硫苷形式稳定存在于植物中。只有当植物组织受到损伤时,萝卜硫苷与黑芥子酶水解反应才生产萝卜硫素。水解过程受到pH,温度,水分等多种因素的影响,从而导致产量的下降。另外,萝卜硫素自身并不稳定,极易挥发,因此从天然植物中提取萝卜硫素具有一定的难度。
西兰花、白菜、甘蓝等十字花科蔬菜被报道富含萝卜硫苷,但经过水煮、爆炒等烹饪处理后,植物源的黑芥子酶会因受热而失去活性,不再具备水解萝卜硫苷的能力。尽管人体的肠道菌群同样具有转化萝卜硫苷为萝卜硫素的能力,但人体对萝卜硫苷存在个体差异。一项人群实验结果显示,志愿者体内对萝卜硫苷的转化率范围为1.1%~40%,但平均转化率仅为10.4%-11.8%。因此,提高肠道菌群对萝卜硫苷的代谢能力,有利于萝卜硫素的吸收与功效的发挥。
炎症常常是许多疾病的起因,Sturm和Wagner将炎症状态与癌症发病风险直接联系起来(NFκB因素)。萝卜硫素是具有最有效的化学预防和良好的抗炎性能的异硫氰酸酯之一。研究表明,萝卜硫素是核转录因子(nuclear factor(erythroid-derived 2)-like 2,Nrf2)的诱导剂,通过激活Nrf2信号通路,可以上调醌氧化还原酶-1(NQO1),超氧化物歧化酶(SOD)等抗氧化酶,从而发挥抗氧化作用。此外,SFN可以通过二硫代氨基甲酸盐的形成与巯基相互作用,抑制氧化还原敏感DNA的结合和NF-κB的反式激活,从而抑制炎症反应的发生。
目前,尚无能够有效转化萝卜硫苷为萝卜硫素,且充分发挥抗炎活性的菌株。因此,期望筛选到一株能够生物转化萝卜硫素的菌株,且萝卜硫素和菌株的协同作用优于单独存在时的效果。
发明内容
本发明要解决的技术问题是提供一株可产萝卜硫素的长双歧杆菌(Bifidobacterium longum)CCFM1206,并将长双歧杆菌CCFM1206单独或与萝卜硫苷(或含萝卜硫苷的组合物)联合用于预防、缓解炎症。
本发明提供了一株长双歧杆菌(Bifidobacterium longum)CCFM1206,已于2021年12月15日保存至广东省微生物菌种保藏中心,保藏编号为GDMCC NO:62129。
所述长双歧杆菌CCFM1206具有如下特性:
(1)形态学特征:细菌体形状不规则,呈弧形、两端大小不一的“V”形或“Y”形。
(2)菌落特征:在MRS固体平板上培养24~48h后,菌落呈光滑、圆形、乳白色或白色不透明,菌落直径为0.5~1mm。
本发明还提供了含有所述长双歧杆菌CCFM1206的益生菌制剂。
在一种实施方式中,所述益生菌制剂中长双歧杆菌CCFM1206的含量≥1×106CFU/g或1×106CFU/mL。
在一种实施方式中,所述益生菌制剂中还含有萝卜硫苷。
在一种实施方式中,所述益生菌制剂中还含有西兰花种子水提物,其中萝卜硫苷含量≥40mg/g。
在一种实施方式中,所述益生菌制剂是利用所述长双歧杆菌CCFM1206的菌液制备的冻干粉剂,其中含有1.0×106cfu/g以上的活性长双歧杆菌CCFM1206。
在一种实施方式中,所述益生菌制剂的制备方法为:将长双歧杆菌CCFM1206以2%~4%的接种量接种于所述MRS培养基中,在37℃条件下厌氧培养24h,离心收集菌体,用pH=7.0~7.2的磷酸盐缓冲液冲洗2-4次,用保护剂重悬达到浓度1010cfu/mL;再将该悬浮液在37℃厌氧条件下培养1h后冷冻干燥制得所述菌剂。
在一种实施方式中,所述保护剂含有100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖、10g/L L谷氨酸钠。
本发明还提供了一种生物转化萝卜硫素的方法,所述方法先将长双歧杆菌CCFM1206接种至发酵培养基中培养至少24h,所述发酵培养基以萝卜硫苷为碳源。
在一种实施方式中,所述发酵培养基含有:蛋白胨10g/L、牛肉膏10g/L、西兰花种子水提物10g/L、无水乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H20 0.1g/L、MnSO4 0.05g/L、吐温-80 1ml/L,半胱氨酸氨酸盐0.5g/L,pH=6.8。
在一种实施方式中,所述西兰花种子水提物含量为10g/L。
本发明还提供了所述长双歧杆菌CCFM1206单独或与萝卜硫苷联合制备预防和/或治疗溃疡性结肠炎(Ulcerative colitis,UC)和/或全身性炎症的药物中的应用。
在一种实施方式中,所述药物还包括药学上可接受的赋型剂;所述药学上可接受的赋型剂是指任何可用于药学领域的稀释剂、辅助剂和/或载体。
本发明还提供了所述长双歧杆菌CCFM1206单独或与含萝卜硫苷的组合物联合制备预防和/或改善溃疡性结肠炎(Ulcerative colitis,UC)和/或全身性炎症症状的产品中的应用。
在一种实施方式中,长双歧杆菌CCFM1206在所述产品中含量不低于1.0×106CFU/mL或1.0×106CFU/g。
在一种实施方式中,所述含萝卜硫苷的组合物是指含有萝卜硫苷的蔬菜或蔬菜提取物;所述蔬菜包括但不限于西兰花、白菜、甘蓝、芥菜中的一种或多种的混合。
在一种实施方式中,所述发酵食品包括发酵牛乳以及乳饮料、发酵果蔬制品;所述果蔬制品包括西兰花、白菜等制成的果汁饮料、果蔬泥、泡菜。
在一种实施方式中,所述改善结溃疡性肠炎症状包括但不限于如下方面:
(1)减少因溃疡性结肠炎而体重减轻的情况;
(2)缓解结肠长度变短的现象;
(3)减少结肠中促炎因子的含量;
(4)调节结肠紧密连接蛋白的转录水平,改善结肠屏障功能。
在一种实施方式中,所述结肠中的促炎因子包括TNF-α,IL-6和IL-1β。
在一种实施方式中,结肠紧密连接蛋白包括Claudin-1,Occudin和ZO-1。
在一种实施方式中,所述改善LPS诱导的全身性炎症症状包括但不限于如下方面:
(1)缓解因炎症引起的脾脏肿大;
(2)降低血清中促炎因子的水平;
(3)减少肝脏组织中促炎因子的含量和提高抗炎因子的含量;
(4)提高短链脂肪酸额含量。
在一种实施方式中,所述血清中促炎因子包括TNF-α,IL-6和IL-1β。
在一种实施方式中,所述肝脏中促炎因子包括TNF-α,IL-6,肝脏抗炎因子包括IL-10。
在一种实施方式中,所述短链脂肪酸包括乙酸、丙酸、丁酸。
有益效果:
(1)本发明筛选出了一株长双歧杆菌(Bifidobacterium longum)CCFM1206,具有萝卜硫素酶活力,可将转化萝卜硫苷转化为萝卜硫素,将此长双歧杆菌CCFM1206接种至改良MRS培养基中发酵24h,在发酵液中检测到16.76μM萝卜硫素。
(2)本发明的长双歧杆菌CCFM1206能够显著促进哺乳动物体内萝卜硫苷的代谢和萝卜硫素的生成,摄于长双歧杆菌CCFM1206可使体内萝卜硫素含量达未摄入情况下的1.5~1.7倍。
(3)本发明提供了一种长双歧杆菌(Bifidobacterium longum)CCFM1206单独或与含萝卜硫苷膳食的组合物联合用于缓解溃疡性结肠炎期间体重的减轻,降低结肠促炎因子的释放,改善结肠屏障功能的用途。
(4)本发明提供了一种长双歧杆菌(Bifidobacterium longum)CCFM1206和含萝卜硫苷膳食的组合物,能够缓解LPS诱导的全身性炎症引起的脾脏肿大,降低血清中促炎因子的水平,减少肝脏组织中促炎因子的含量和提高抗炎因子的含量,提高短链脂肪酸的含量。
生物材料保藏
一株长双歧杆菌(Bifidobacterium longum)CCFM1206,分类学名为Bifidobacterium longum,已于2021年12月15日保存至广东省微生物菌种保藏中心,保藏编号为GDMCC NO:62129,保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1是长双歧杆菌(Bifidobacterium longum)CCFM1206的菌落形态图;
图2是不同样品的萝卜硫素含量的检测结果;
图3是不同处理组UC小鼠体重变化图;
图4是不同处理组UC小鼠结肠长度变化图;
图5是不同处理组UC小鼠结肠组织形态(HE染色);
图6是不同处理组UC小鼠结肠中促炎因子含量图;
图7是不同处理组UC小鼠结肠紧密连接蛋白转录水平图;
图8是不同处理组LPS小鼠脾脏指数图;
图9是不同处理组LPS小鼠血清中促炎因子含量图;
图10是不同处理组LPS小鼠肝脏中炎症相关因子含量图;
图11是不同处理组LPS小鼠粪便中短链脂肪酸含量。
图12是不同处理组UC小鼠和LPS小鼠粪便中萝卜硫素含量。
图13是普通饲料小鼠和含萝卜硫苷膳食饲料小鼠粪便中萝卜硫素含量。
具体实施方式
下述实施例中涉及SPF级6周龄雄性C57BL/J小鼠购于维通利华实验动物有限公司;下述实施例中涉及的葡聚糖硫酸钠盐(Dextran Sulfate Sodium Salt,DSS),脂多糖(Lipopolysaccharide,LPS)购于上海sigma公司;西兰花种子水提物购于赣州华汉生物科技有限公司,每g西兰花种子水提物中萝卜硫苷含量(按质量百分比计)为20%;下述实施例中涉及的ELISA试剂盒购于上海酶联生物科技有限公司;下述实施例中涉及的其他试剂购于国药集团化学试剂有限公司。
下述实施例中涉及的培养基如下:
MRS固体培养基:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、琼脂20g/L、半胱氨酸氨酸盐0.5g/L。
MRS液体培养基:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
改良MRS固体培养基:蛋白胨10g/L、牛肉膏10g/L、西兰花种子水提物10g/L、无水乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H20 0.1g/L、MnSO4 0.05g/L、吐温-80 1ml/L、琼脂20g/L、半胱氨酸氨酸盐0.5g/L。
改良MRS液体培养基:蛋白胨10g/L、牛肉膏10g/L、西兰花种子水提物10g/L、无水乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2PO4·3H2O 2.6g/L、MgSO4·7H20 0.1g/L、MnSO4 0.05g/L、吐温-80 1ml/L、半胱氨酸氨酸盐0.5g/L。
下述实施例中涉及的检测方法如下:
萝卜硫素的检测方法:使用UPLC-Q Exactive四极杆-静电场轨道阱高分辨质谱仪(Thermo Fisher Scientific公司,美国)的二级平行监测(parallet-reaction-monitoting,PRM)进行定性、定量分析。色谱柱为Waters HSS T3色谱柱(1.8μm×2.1mm×100mm);柱温为35℃;流动相为:A-0.1%甲酸水,B-乙腈;流速为0.3min/L;进样量为2μL;梯度洗脱:0-3.0min 5%B,3-9min 5%-30%B,9-15min 30%-100%B,15-16min 100%B,16-16.5min 100%-5%B,16.5-20min 5%B。离子源采用HESI源(heated ESI),喷雾电压:3.5kV(+)、3.2kV(-),鞘气体积流量:35μL·min-1,离子传输管温度:320℃;辅助气体流量:15μL·min-1;辅助气温度:320℃。扫描模式:PRM(100-500m/z);分辨率:35000;采集极性:positive;AGC target:5e 5;Maximun IT:100ms。
短链脂肪酸含量的测定:使用Trace 1300GC-MS气质谱联用(Thermo FisherScientific公司,美国)进行测定。色谱柱为Rtx Wax柱(柱长30m,内径25μm);载气为氦气,流速为2mL/min;进样体积1μL,按7.5℃/min升温至140℃,然后按60℃/min升温至200℃保持3min,离子化温度20℃;分析采用全扫描模式,为通过外标法值得标准曲线,从而计算出各短链脂肪酸浓度。
实施例1:长双歧杆菌CCFM1206的筛选、鉴定、观察和保存。
1、筛选
取0.5g来自健康成人的新鲜粪便样品添加至4.5mL 0.9%生理盐水中进行梯度稀释,选择合适的梯度稀释液涂布在添加了0.2%溴甲酚紫的MRS固体改良培养基中,置于37℃厌氧条件下培养24~48h。选取变色圈明显变黄的单菌落接种至MRS平板上划线纯化,挑取单菌落转接至液体MRS液体培养基增菌,30%甘油保藏,得到菌株长双歧杆菌CCFM1206。
2、鉴定
提取菌株CCFM1206的全基因组DNA用于16S rDNA的扩增,收集扩增后的DNA片段用和测序(由苏州金唯智生物科技有限公司完成),将该序列在NCBI中进行核酸序列比对,结果显示菌株为长双歧杆菌,命名为长双歧杆菌(Bifidobacterium longum)CCFM1206。
3、观察
蘸取长双歧杆菌(Bifidobacterium longum)CCFM1206的菌液在在MRS固体培养基上划线,于37℃厌氧培养48h后,观察其菌落,发现其菌落呈圆形、白色、光滑(图1)。
4、保存
挑取长双歧杆菌(Bifidobacterium longum)CCFM1206的单菌落接种于MRS液体培养基中,37℃厌氧培养24h,得到菌液;取1mL菌液于无菌离心管中,8000r/min离心3min后弃去上层培养基,菌泥重悬于30%甘油溶液中置于-80℃中保藏。
实施例2:长双歧杆菌CCFM1206发酵生产萝卜硫素
将-80℃保藏的长双歧杆菌CCFM1206划线于MRS固体培养基中,在37℃厌氧培养24~48h,再经MRS液体培养基传代2~3次后,以2%~4%的接种量接种于改良MRS液体培养基中,于37℃厌氧培养24h,得到的发酵液用于萝卜硫素含量检测。
结果如图2所示。接种后的培养基中不含萝卜硫素,经过长双歧杆菌CCFM1206发酵24h后可检测到萝卜硫素含量为16.76μM。
实施例3:长双歧杆菌CCFM1206和含萝卜硫苷膳食复配对UC小鼠疾病症状的影响:
取6周龄的健康雄性C57BL/6J小鼠40只,适应环境一周后,随机分为5组,每组8只小鼠。5组分别为空白组、模型组、含萝卜硫苷膳食组(BSE)、长双歧杆菌CCFM1206组(CCFM1206)以及含萝卜硫苷膳食复配长双歧杆菌CCFM1206组(BSE+CCFM1206)。
第8-21天为14天灌胃干预期,每次灌胃剂量均为0.2mL/只,每天灌胃时间一致。其中,对照组和模型组灌胃生理盐水,BSE组灌胃40mg/mL的西兰花种子水提物溶液,CCFM1206组灌胃含有5×109CFU/mL的菌悬液,BSE+CCFM1206组灌胃含有40mg/mL的西兰花种子水提物和5×109CFU/mL菌悬液的混合物。
第15-21天为干预期的最后7天,进行溃疡性结肠炎造模。在饮水中添加浓度为2.5%(w/v)的DSS。第22天牺牲小鼠,收集血清、组织等用于相关指标测定。实验动物分组及处理方式见表1。
表1动物分组及处理方法
造模期间(DSS处理期间),每天定时称量小鼠体重并计算其体重变化百分比。牺牲小鼠后,测量小鼠结肠长度,计算每组小鼠结肠平均长度。实验结果如图3、图4所示,模型组小鼠体重从第19天出现显著下降,并在第21天体重下降超了10%,此外模型组小鼠平均结肠长度(5.57±0.33)显著低于空白组(6.87±0.48)。而BSE组,CCFM1206组以及CCFM1206+BSE组小鼠体重下降和结肠变短均被明显缓解。其中,CCFM1206+BSE组小鼠平均结肠长度(6.46±0.58)略高于BSE组(6.34±0.55)和CCFM1206组(6.27±0.35)。这些实验结果说明BSE、CCFM1206、CCFM1206+BSE对缓解结肠炎小鼠的疾病症状有效。
实施例4:长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物可改善UC小鼠结肠粘膜损伤
C57BL/6J小鼠分组、造模及处理方法同实施例3。
在第22天牺牲小鼠,收集小鼠结肠组织,制作小鼠结肠石蜡切片,具体实验步骤为:取距***1cm处的一段远端结肠1cm,用4%多聚甲醛固定48h。将固定好的结肠组织用流水冲洗8h后,依次放入70%、80%、90%乙醇溶液进行脱水,每次脱水30min,再依次放入95%、100%的乙醇溶液,每次20min。将结肠样品置于1:1的二甲苯和酒精混合液中15min,然后放入二甲苯I和二甲苯II各15min。将结肠组织转移至二甲苯和石蜡各半的混合液15min,再放入石蜡Ⅰ、石蜡Ⅱ透蜡各1h,温度保持60℃。用莱卡石蜡包埋机将结肠包埋于重新融化的蜡块中,用组织切片机对包埋好的组织切片,厚度为5μm。粘片后晾干,置于62℃烘箱中1h。
石蜡切片制作完成后,进行HE染色,具体实验步骤为:石蜡切片经二甲苯Ⅰ、Ⅱ脱蜡各5min,在依次放入100%、95%、90%、80%、70%乙醇溶液中各3 5min,最后放入蒸馏水中3min。用苏木精染色20s,用蒸馏水洗去未结合的苏木精。再用伊红染色2s,依次进入95%乙醇Ⅰ、Ⅱ,70%乙醇中,并快速拿出,然后进入80%乙醇中50~55s,进入无水乙醇中2min。切片放入1:1混合的二甲苯-酒精混合液中1min,接着放入二甲苯Ⅰ、Ⅱ中各2~3min,用中性树胶封片。
结果如图5所示,模型组小鼠的结肠上出现大量炎性细胞浸润,大量黏膜上皮细胞变性、坏死,杯状细胞数量明显减少、隐窝消失,以及组织水肿等病理现象。灌胃BSE和长双歧杆菌CCFM1206虽然对结肠炎有一定程度的缓解,但是结肠组织出现水肿、大量炎症细胞的浸润以及一些杯状细胞减少。而CCFM1206+BSE组的小鼠结肠炎明显得到了改善,与空白组相比,除炎性细胞明显增多外,其他结构基本保持完整。说明长双歧乳杆菌CCFM1206和含萝卜硫苷膳食复配能够显著改善UC小鼠结肠粘膜损伤,且效果优于单独灌胃长双歧杆菌CCFM1206或西兰花种子水提物。
实施例5:长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物可显著降低结肠炎中促炎因子含量
C57BL/6J小鼠分组、造模及处理方法同实施例3。
在第22天牺牲小鼠,收集小鼠结肠组织。小鼠结肠组织按1:9比例加入入预冷PBS缓冲液进行组织研磨,12000g,离心15min,取上清,分别按照TNFα、IL 1β、IL 6酶联免疫试剂盒的检测方法测定结肠中TNFα、IL 1β和IL 6的含量。
实验结果如图6所示,模型组组小鼠结肠中促炎因子TNF-α、IL-6和IL-1β含量显著增高,而BSE组、CCFM1206组、CCFM1206+BSE组可将TNF-α由模型组的66.53±6.12分别降低至55.42±9.72、59.13±5.68、47.45±8.04;将IL-6由模型组的10.36±1.37分别降低至6.54±0.61、8.26±0.89、6.10±1.35;将IL-1β由模型组的5.53±0.43分别降低至4.38±0.79、4.31±0.66、3.91±0.76;都能显著降低这些炎症因子的水平,但是长双歧CCFM1206和西蓝花种子水提物组合物效果最好,炎症因子下降水平最显著。
实施例6:长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物可增强肠道屏障功能
C57BL/6J小鼠分组、造模及处理方法同实施例3。
在第22天牺牲小鼠,收集小鼠结肠组织,测定结肠中紧密连接相关蛋白Claudin-1、Occudin和ZO-1的转录水平。
测定方法如下:合成Claudin-1、Occudin、ZO-1和GAPDH的引物序列,引物信息如表3。取小鼠同部位的结肠组织1cm,迅速放入液氮中,置于负80℃冰箱冻存,取出冻存结肠组织放入已加入1mL TRIzol和3粒锆珠的1.5mL无酶离心管中,用组织研磨匀浆机充分匀浆,室温静置5min。加入0.2mL氯仿,剧烈振荡30s,静置10min。接着以12000g,4℃离心15min。小心吸取上层水相至一新的无酶1.5mL离心管中,加入等体积异丙醇,上下轻轻颠倒混匀,室温静置10min。接着以12000g,4℃离心15min。弃去上清,加入1mL预冷75%乙醇,轻弹洗涤沉淀。以12000g,4℃离心5min,小心吸取弃去上清,于超净台内吹干沉淀,加入50μL无酶超纯水溶解RNA。使用微量分光光度计测定提取的RNA浓度,OD260/OD280在1.9~2.0之间的质量合格。以提取质量合格的总RNA为模板,按照反转录试剂盒说明书的步骤合成cDNA。反转录得到的cDNA进行qRT PCR检测,PCR体系为:5μLSYBR Green Supermix,3μL去离子水,0.5μL上游引物(10μmol/L),0.5μL下游引物(10μmol/L)和1μL的cDNA模板(100ng/μL)。qPCR运行程序设定:94℃,2min,(94℃,30s;61℃,30s;72℃,20s)39个循环;目的基因经过Real timePCR检测后,以GAPDH为内参基因,采用2△△CT法进行相对基因表达分析。
表2引物序列
实验结果如图7所示,模型组小鼠结肠中三种紧密连接蛋白Claudin-1、Occudin、ZO-1的mRNA表达水平显著下降,而BSE组、CCFM1206组CCFM1206+BSE组都上调了这些紧密连接蛋白的表达水平。其中,BSE组、CCFM1206组CCFM1206+BSE组可将Claudin-1由模型组的E1.00±0.11分别提高至1.94±0.21、2.86±0.50、3.90±0.56;将Occudin由模型组的1.01±0.17分别提高至2.16±0.32、3.12±0.31、4.51±0.71,将ZO-1由模型组的1.03±0.29分别提高至2.08±0.51、2.91±0.34、3.23±0.67。
实施例7:长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物可降低LPS小鼠脾脏指数
取6周龄的健康雄性C57BL/6J小鼠40只,适应环境一周后,随机分为5组,每组8只小鼠。5组分别为空白组、模型组、含萝卜硫苷膳食组(BSE)、长双歧杆菌CCFM1206组(CCFM1206)以及含长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物组(BSE+CCFM1206)。
第8-21天为14天灌胃干预期,每次灌胃剂量均为0.2mL/只,每天灌胃时间一致。其中,对照组和模型组灌胃生理盐水,BSE组灌胃40mg/mL的西兰花种子水提物溶液,CCFM1206组灌胃含有5×109cfu/mL的菌悬液,BSE+CCFM1206组灌胃含有40mg/mL的西兰花种子水提物和5×109cfu/mL菌悬液的混合物。
第22天对小鼠进行腹腔注射,空白组小鼠注射0.9%生理盐水,其他组小鼠注射6mg/kg的LPS,4小时后对小鼠进行称重,牺牲小鼠,收集血清、组织等用于相关指标测定。实验动物分组及处理方式见表3。
表3动物分组及处理方法
LPS诱导的全身性炎症会引起小鼠脾脏肿大。对小鼠脾脏进行称重,计算脾脏指数。
实验结果如图8所示,模型组小鼠脾脏指数由空白组的0.25±0.02升高至0.34±0.01,说明模型组小鼠脾脏因LPS造成的肿大较为明显,而灌胃长双歧杆菌CCFM1206(0.34±0.02)对缓解脾脏肿大的效果并不明显,灌胃含萝卜硫苷膳食以及长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物与对照组相比分别显著降低至0.32±0.02和0.31±0.02,且长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物的效果优于单独灌胃含萝卜硫苷膳食。说明长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物能缓解因炎症引起的脾脏肿大。
实施例8:长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物可降低LPS小鼠血清中促炎因子含量
C57BL/6J小鼠分组、造模及处理方法同实施例7。
第22天,小鼠通过眼球取血得到血浆,3500g离心15min,取上清得到血清。分别按照TNFα、IL 1β、IL 6酶联免疫试剂盒的检测方法测定血清中促炎因子的含量。
实验结果如图9所示,模型组小鼠血清中TNFα、IL 1β、IL 6含量分别显著增加至109.39±10.71、10.20±1.92、9.98±0.97,而BSE组、CCFM1206组和CCFM1206+BSE组的小鼠血清中促炎因子TNFα含量可分别下降至97.03±10.51、93.91±10.49和88.87±11.23,促炎因子IL-1β含量分别下降至7.52±1.18、9.34±1.23和6.43±1.83。BSE组、CCFM1206组促炎因子IL-6变化并不明显(9.34±1.44、10.63±1.09),而CCFM1206+BSE组的小鼠血清中促炎因子IL-6含量可下降至8.68±1.64。说明长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物能够缓解小鼠因LPS引起的全身性炎症。
实施例9:长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物可降低LPS小鼠肝脏中炎症因子含量
C57BL/6J小鼠分组、造模及处理方法同实施例7。
第22天,牺牲小鼠后解剖得到小鼠肝脏。小鼠肝脏组织按1:9比例加入预冷PBS缓冲液进行组织研磨,12000g,离心15min,取上清,分别按照TNFα、IL 6和IL 10酶联免疫试剂盒的检测方法测定结肠中TNFα、IL 6和IL 10的含量。
实验结果如图10所示,模型组小鼠肝脏中促炎因子TNFα和IL 6含量显著增加至31.10±2.33和58.07±4.42,抗炎因子IL-10显著降低至80.89±5.12;灌胃长双歧杆菌CCFM1206对降低促炎因子TNF-α和IL-6(30.59±1.99、53.62±5.59)效果不显著,但能显著提高抗炎因子IL-10的水平(105.10±8.40);而灌胃含萝卜硫苷膳食以及长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物的小鼠肝脏中促炎因子TNFα含量显著下降至27.92和25.51±2.03,促炎因子IL 6含量显著下降至49.41±5.00和48.88±4.58,抗炎因子IL-10含量显著增加至93.82±8.76和110.12±5.79,说明长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物能够有效缓解小鼠因LPS引起的全身性炎症。
实施例10:长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物可增加LPS小鼠粪便中短链脂肪酸含量
C57BL/6J小鼠分组、造模及处理方法同实施例7。
第22天牺牲小鼠后,收集小鼠结肠内容物冻存于-80℃。首先对粪便进行冻干处理,称取50mg粪便,用500μL饱和NaCl溶液重悬,加入20μL的10%H2 SO4酸化;加入800μL无水***,震荡均匀,提取脂肪酸,然后13000g离心15min;取上层***相,加入0.25g无水Na2SO4进行干燥;振荡混匀30s后13000g离心10min取上层***相,利用GC-MS测定小鼠冻干粪便中的短链脂肪酸乙酸、丙酸、丁酸含量。
实验结果如图11所示,与空白组(57.68±10.17、21.95±4.42、4.63±0.68)相比,模型组小鼠粪便中乙酸、丙酸、丁酸的含量显著下降至28.68±4.4、8.83±1.65、2.03±0.45,而灌胃长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物的小鼠粪便中乙酸含量显著增加至42.95±9.14,丙酸含量显著增加至14.44±1.83,丁酸含量显著增加至3.35±0.56,且效果优于单独灌胃含萝卜硫苷膳食(33.37±7.42、10.88±2.45/2.43±0.34)或长双歧杆菌CCFM1206(37.46±6.17、10.98±2.46、2.91±0.58)。
实施例11:长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物可增加小鼠体内代谢萝卜硫素含量
C57BL/6J小鼠分组、造模及处理方法同实施例3、7。
第22天牺牲小鼠后,收集小鼠结肠内容物冻存于-80℃。称取100mg内容物加入800μL甲醇沉淀蛋白,加入2-3粒镐珠60Hz研磨5min,4℃13000g离心15min。取400μL上清液蒸发至干,再加200μL甲醇-水(4:1)复溶,4℃13000g离心15min,取上清过0.22μm滤膜后进行测定。
实验结果如图12所示,未灌胃含萝卜硫苷膳食的组别小鼠内容物中检测不到萝卜硫素,而灌胃长双歧乳杆菌CCFM1206和含萝卜硫苷膳食组合物显著增加了UC小鼠和LPS小鼠结肠内容物中萝卜硫素的含量,分别为灌胃含萝卜硫苷膳食的1.5倍和1.7倍。说明长双歧杆菌CCFM1206能够提高体内萝卜硫素产量。
实施例12:长双歧杆菌CCFM1206可促进膳食中萝卜硫苷转化为萝卜硫素。
取6周龄的健康雄性C57BL/6J小鼠24只,适应环境一周后,随机分为3组,每组8只小鼠。3组分别为正常饮食组、含萝卜硫苷膳食饲料组、以及含萝卜硫苷膳食饲料加灌胃长双歧杆菌CCFM1206组(复合组)。
所述含萝卜硫苷膳食饲料为在正常市售小鼠饲料中额外添加了含萝卜硫苷的蔬菜冻干粉或蔬菜提取物制成的饲料,其中每克饲料中含有0.6mg的萝卜硫苷。所述蔬菜包括但不限于西兰花、白菜、甘蓝等蔬菜的混合物。
第8-14天为7天灌胃干预期,每次灌胃剂量均为0.2mL/只,每天灌胃时间一致,小鼠采食量约3g/日。其中,正常饮食组和含萝卜硫苷膳食饲料组灌胃生理盐水,复合组灌胃含有5×109cfu/mL的长双歧杆菌CCFM1206菌悬液。在第15天牺牲小鼠,收集小鼠结肠内容物冻存于-80℃。称取100mg内容物加入800μL甲醇沉淀蛋白,加入2-3粒镐珠60Hz研磨5min,4℃13000g离心15min。取400μL上清液蒸发至干,再加200μL甲醇-水(4:1)复溶,4℃13000g离心15min,取上清过0.22μm滤膜后进行测定萝卜硫素含量。实验动物分组及处理方式见表4。
表4动物分组及处理方法
实验结果如图13所示,正常饮食组小鼠内容物中未检测到萝卜硫素,而给予含萝卜硫苷膳食饲料的小鼠内容物中均有萝卜硫素检出,且复合组中在长双歧杆菌CCFM1206的作用下小鼠结肠内容物中萝卜硫素含量显著增高,约为含萝卜硫苷膳食饲料组的1.7倍。说明长双歧杆菌CCFM1206可促进膳食中萝卜硫苷转化为萝卜硫素。
实施例13:长双歧杆菌CCFM1206和含萝卜硫苷膳食组合物的制备。
培养基的配制:蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K2 PO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4 0.05g/L、吐温801mL/L、半胱氨酸氨酸盐0.5g/L,pH为6.8。
冻干保护剂的制备:使用水与保护剂原料混合制备得到含有100g/L脱脂奶粉、30mL/L甘油、100g/L麦芽糊精、150g/L海藻糖、10g/L L谷氨酸钠的保护剂。
将长双歧杆菌CCFM1206以2%~4%的接种量接种于所述MRS培养基中,在37℃条件下厌氧培养24h,离心收集菌体,用pH=7.0~7.2的磷酸盐缓冲液冲洗2-4次,用所述的保护剂重悬达到浓度1010cfu/mL;再将该悬浮液在37℃厌氧条件下培养1h后冷冻干燥制得长双歧杆菌CCFM1206菌剂。
可选地,将所制菌剂与西兰花种子水提物混合,保证组合物中长双歧杆菌CCFM1206活菌数不低于1.0×106CFU/mL或1.0×106CFU/g,西兰花种子水提物含量不低于200mg/g。
可选地,可将所述鼠李糖乳杆菌CCFM1252菌剂与含萝卜硫苷的蔬菜或蔬菜提取物共同制备膳食补充剂;所述蔬菜包括但不限于西兰花、白菜、甘蓝、芥菜中的一种或多种的混合。
可选地,还可以利用长双歧杆菌CCFM1206制备功能性菌制剂、发酵食物、或药物组合物。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (12)
1.一株长双歧杆菌(Bifidobacterium longum)CCFM1206,已于2021年12月15日保存至广东省微生物菌种保藏中心,保藏编号为 GDMCC NO:62129。
2.含有权利要求1所述长双歧杆菌CCFM1206的益生菌制剂。
3.根据权利要求2所述的益生菌制剂,其特征在于,所述益生菌制剂中长双歧杆菌CCFM1206的含量≥1×106CFU/g或1×106CFU/mL。
4.根据权利要求3所述的益生菌制剂,其特征在于,所述益生菌制剂中还含有萝卜硫苷或西兰花种子水提物。
5.含有权利要求1所述长双歧杆菌CCFM1206的食品。
6.含有权利要求1所述长双歧杆菌CCFM1206的保健品。
7.含有权利要求1所述长双歧杆菌CCFM1206的膳食补充剂。
8.根据权利要求5所述的食品,或权利要求6所述的保健品,或权利要求7所述的膳食补充剂,其特征在于,还含有含萝卜硫苷的组合物;所述含萝卜硫苷的组合物是指含有萝卜硫苷的蔬菜或蔬菜提取物;所述蔬菜为西兰花、白菜、甘蓝、芥菜中的一种或多种的混合。
9.含有权利要求1所述长双歧杆菌CCFM1206的药物,其特征在于,长双歧杆菌CCFM1206的含量≥1×106CFU/g或1×106CFU/mL。
10.权利要求1所述的长双歧杆菌CCFM1206单独或与萝卜硫苷联合制备预防和/或治疗溃疡性结肠炎、全身性炎症的药物中的应用。
11.权利要求1所述的长双歧杆菌CCFM1206在制备发酵食品中的应用。
12.权利要求1所述的长双歧杆菌CCFM1206在制备保健品中的应用。
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