CN114752523B - 一株代谢产生萝卜硫素减轻炎症反应的鼠李糖乳杆菌ccfm1252 - Google Patents
一株代谢产生萝卜硫素减轻炎症反应的鼠李糖乳杆菌ccfm1252 Download PDFInfo
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Abstract
本发明公开了一株代谢产生萝卜硫素减轻炎症反应的鼠李糖乳杆菌CCFM1252,属于微生物技术领域。本发明提供了一株具有较强的萝卜硫苷代谢能力,并能生物转化萝卜硫素的鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252,本发明提供的鼠李糖乳杆菌CCFM1252可单独或与萝卜硫素的联合用于改善溃疡性结肠炎和/或全身性炎症症状,可缓解溃疡性结肠炎期间体重的减轻,黏膜损伤,降低结肠氧化应激水平,调节结肠促炎因子转录水平,减少促炎因子的释放;并具有降低氧化应激水平,减少促炎因子的释放,缓解结肠、肝脏等损伤的潜力。
Description
技术领域
本发明涉及一株代谢产生萝卜硫素减轻炎症反应的鼠李糖乳杆菌CCFM1252,属于微生物技术领域。
背景技术
萝卜硫素是一种异硫氰酸酯,可由萝卜硫苷经黑芥子酶水解而得,分子式为为C6H11NOS2。萝卜硫素通常并不天然存在于植物中,而是以前体萝卜硫苷的形式存在十字花科植物中,而且当植物组织受到损伤时,萝卜硫苷与黑芥子酶反应生产萝卜硫素。但是萝卜硫素自身不稳定,易挥发,在高温和碱性条件下易分解,因此,萝卜硫素的提取存在一定的困难。
萝卜硫素因其一些抗炎、抗癌、抗氧化等功效,而受到人们的广泛关注。研究表明,萝卜硫素通过抑制抑制促炎NF-κB信号通路,可以抑制促炎因子(例如IL-6,TNF-α,IL-1β),从而缓解炎症相关疾病。此外,萝卜硫素是核转录因子(nuclear factor(erythroid-derived 2)-like2,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。
人体的肠道细菌能够代谢西兰花、白菜、甘蓝等十字花科蔬菜中的萝卜硫苷为萝卜硫素,但是对萝卜硫苷的代谢吸收效率并不高,且个体差异较大。研究表明,志愿者在使用含萝卜硫苷制剂干预后,尿液中的萝卜硫素排泄通常为剂量的2%~15%;如果食用萝卜硫素制剂,尿液中的萝卜硫素排泄为剂量的70~90%。因此,促进肠道菌群对萝卜硫素的转化或是利用肠道细菌预先生成萝卜硫素,有利于萝卜硫素的吸收与功能的发挥。
目前,尚无能够代谢产生萝卜硫素减轻炎症反应的菌株。因此,期望筛选到一株能够将植物源萝卜硫苷代谢为萝卜硫素的菌株。
发明内容
本发明要解决的技术问题是提供一株能够代谢萝卜硫苷产生萝卜硫素的鼠李糖乳杆菌CCFM1252(Lacticaseibacillus rhamnosus),并将鼠李糖乳杆菌CCFM1252和萝卜硫苷联合作用于缓解炎症。
本发明提供了一株鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252,已于2022年3月6日保存至广东省微生物菌种保藏中心,保藏编号为GDMCC NO:62273。
所述鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252具有如下特征:
(1)细菌菌形态呈双杆状,短小的杆菌,不运动,无芽孢,为革兰氏阳性菌。
(2)在MRS固体平板上有氧或厌氧培养24~48h后,菌落呈凸起、乳白色、面湿润光滑、边缘不齐的圆形,直径在0.5~2mm之间。
本发明还提供了含有所述鼠李糖乳杆菌CCFM1252的益生菌制剂。
在一种实施方式中,所述益生菌制剂中鼠李糖乳杆菌CCFM1252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西兰花种子水提物。
在一种实施方式中,所述菌剂的制备方法为:将鼠李糖乳杆菌CCFM1252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后冷冻干燥制得所述菌剂。
在一种实施方式中,冻干保护剂是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。
在一种实施方式中,所述益生菌制剂中还加入有≥200mg/g西兰花种子水提物。
本发明提供了所述鼠李糖杆菌CCFM1252单独或与萝卜硫苷联合制备预防和/或改善溃疡性结肠炎(Ulcerative colitis,UC)、全身性炎症症状的功能性产品中的应用。
在一种实施方式中,所述产品包括能够治疗/改善溃疡性结肠炎和或全身性炎症症状的药物。
在一种实施方式中,所述功能性产品中鼠李糖乳杆菌CCFM1252的含量不低于1.0×106CFU/mL或1.0×106CFU/g。
在一种实施方式中,所述食品、保健品或药物中含有鼠李糖乳杆菌CCFM1252和含量≥200mg/g西兰花种子水提物。
在一种实施方式中,所述食品为发酵食品;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,所述药物或药物组合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。
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状况;
(2)缓解结肠黏膜损伤;
(3)降低结肠氧化应激水平;
(4)调节结肠促炎因子转录水平,减少促炎因子的释放。
在一种实施方式中,所述结肠中的氧化应激水平指标包括丙二醛(MDA)含量,总抗氧化能力(T-AOC),髓过氧化物酶(MPO)含量。
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)缓解血清中促炎因子,的水平
(3)降低肝脏组织中AST/ALT比率,缓解肝功能损伤
(4)降低肝脏组织中氧化应激水平
在一种实施方式中,所述血清中的促炎因子包括TNF-α,IL-6和IL-1β。
在一种实施方式中,所述肝脏组织中AST/ALT比率指谷草转氨酶/谷丙转氨酶酶活力比值。
在一种实施方式中,所述肝脏组织中的氧化应激水平指标包括丙二醛(MDA)含量,超氧化物歧化酶(SOD)酶活力以及谷胱甘肽过氧化物酶(GSH-Px)酶活力。
在一种实施方式中,所述含萝卜硫苷膳食是指含有萝卜硫苷的蔬菜,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。
本发明还提供所述鼠李糖乳杆菌CCFM1252在发酵领域的应用。
有益效果:
(1)本发明筛选出了一株鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252,具有较强的代谢萝卜硫苷的能力,并具有萝卜硫素酶活力,酶活力为2.61×10- 4U/mg蛋白,可将萝卜硫苷转化为萝卜硫素。
(2)本发明提供了一种鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252,日常补充该菌株可将膳食中进入大肠的萝卜硫苷高效代谢转化为萝卜硫素,具有降低氧化应激水平,减少促炎因子的释放,缓解结肠、肝脏等损伤的潜力。
(3)本发明将鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252单独或/和含萝卜硫苷膳食的组合物联合用于溃疡性结肠炎小鼠,可缓解溃疡性结肠炎期间体重的减轻,黏膜损伤,降低结肠氧化应激水平,并调节结肠促炎因子转录水平,减少促炎因子的释放。且鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252和含萝卜硫苷膳食的组合物在缓解溃疡性结肠炎的效果优于单独含萝卜硫苷膳食和单独菌株。
(4)本发明将鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252单独或/和含萝卜硫苷膳食的组合物联合用于LPS诱导的全身性炎症小鼠,可缓解因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,降低肝脏组织中AST/ALT比率,缓解肝功能损伤,降低肝脏组织中氧化应激水平,提高抗氧化作用。且鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252和含萝卜硫苷膳食组合物在缓解LPS诱导的全身性炎症的效果优于单独含萝卜硫苷膳食和单独菌株。
生物材料保藏
一株鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252,分类学名为Lacticaseibacillus rhamnosus,已于2022年3月6日保存至广东省微生物菌种保藏中心,保藏编号为GDMCC NO:62273,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是鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252的菌落形态图;
图2是鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252在改良MRS中的生长曲线图;
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小鼠结肠组织形态(HE染色);
图5是不同处理组UC小鼠结肠氧化应激水平图;
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小鼠结肠中促炎因子转录水平图;
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AST/ALT比率图;
图11是不同处理组LPS小鼠肝脏中氧化应激水平图。
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;下述实施例中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:
萝卜硫苷的检测方法:在Waters 1525HPLC系统中使用紫外(UV))检测器(229nm)分析,使用AQ-C18色谱柱(5μm×4.6mm×150mm)进行分离,在30℃下等度洗脱,流速为1.0mL/min,流动相由95%0.08mol/L磷酸二氢钠溶液和5%甲醇(v/v)。
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。
实施例1:鼠李糖乳杆菌CCFM1252的筛选、鉴定、观察和保存。
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,得到菌株鼠李糖乳杆菌CCFM1252。
提取菌株CCFM1252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,结果显示菌株为鼠李糖乳杆菌,命名为鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252。
蘸取鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252的菌液在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、乳白色、光滑(具体可见图1)。
实施例2:鼠李糖乳杆菌CCFM1252在含萝卜硫苷的培养基中的生长情况
将冷冻保存的鼠李糖杆菌CCFM1252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℃厌氧环境中静置培养,每隔2h测定培养液的OD600值,并保留上清液用于HPLC检测萝卜硫苷含量。待鼠李糖CCFM1252生长进入稳定期,停止测定。
实验结果如图2所示,鼠李糖乳杆菌CCFM1252在含有萝卜硫苷的改良MRS培养基中,OD600值不断增加,并在10h左右进入稳定期。HPLC检测萝卜硫苷含量发现,萝卜硫苷含量随生长时间的延长不断下降,且变化趋势与鼠李糖乳杆菌CCFM1252的OD600值基本一致,说明鼠李糖乳杆菌CCFM1252在生长过程中代谢了萝卜硫苷。
实施例3:鼠李糖乳杆菌CCFM1252代谢萝卜硫素酶活力的测定
将冷冻保存的鼠李糖杆菌CCFM1252划线于MRS固体培养基中,在37℃厌氧培养24~48h,再活化2~3次后,以2%~4%的接种量接种于MRS液体培养基中于37℃厌氧环境下静置培养24h后,取1mL菌液离心收集菌泥,用0.05mM的磷酸盐缓冲溶液(pH=7.0)重悬菌泥,用45Hz超声破碎后,离心得到细胞破碎上清液。于1.5mL离心管中加入100μL浓度为1mM的萝卜硫苷,600μL细胞破碎上清液,100μL 1mM抗坏血酸,其中萝卜硫苷和抗坏血酸均用磷酸盐缓冲液溶解。在37℃反应150min后,保留上清液用于萝卜硫素含量和蛋白含量的测定。空白对照以600μL磷酸盐缓冲液代替细胞破碎上清液。定义:37℃下,每分钟转化1μmol萝卜硫素的酶量(mg)为1个酶活力单位。
实验结果测得反应后上清液中萝卜硫素含量为3.46mg/L,测得细胞破碎上清液中蛋白含量为0.665mg/mL,计算的酶活力为2.61×10-4U/mg。且空白对照中未检测到萝卜硫素的生成,说明鼠李糖乳杆菌CCFM1252能够代谢萝卜硫苷生成萝卜硫素。
实施例4:鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物缓解UC小鼠体重下降
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)、鼠李糖乳杆菌组(CCFM1252)以及鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物组(BSE+CCFM1252)。
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,CCFM1252组灌胃含有5×109cfu/mL的菌悬液,BSE+CCFM1252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。模型组小鼠在DSS处理下,体重显著下降,而BSE组、CCFM1252组以及BSE+CCFM1252组小鼠体重下降的趋势明显被缓解,说明鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物能够缓解DSS引起的小鼠体重下降。
实施例5:鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物改善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4。
在第22天牺牲小鼠,收集小鼠结肠组织,生理盐水轻微漂洗后置于4%多聚甲醛固定液中固定。固定48h后进行石蜡包埋、切片,采用苏木精-伊红染色(HE),观察结肠绒毛等组织形态。
结果如图4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。BSE和CCFM152组小鼠结肠组织仍出现出现水肿、大量炎症细胞的浸润以及一些杯状细胞减少。而BSE+CCFM1252组的小鼠结肠炎明显得到了改善,除少量炎性细胞浸润外,其他结构基本保持完整,整体组织形态与空白组小鼠结肠接近。说明鼠李糖乳杆菌CCFM1252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鼠李糖杆菌CCFM1252或含萝卜硫苷膳食。
实施例6:鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物降低UC小鼠结肠氧化应激水平
C57BL/6J小鼠分组、造模及处理方法同实施例4。
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MDA、SOD、GSH试剂盒的检测方法测定结肠中MDA、T-AOC以及MPO。
实验结果如图5所示,相比于空白组(MDA、SOD、GSH分别为1.55±0.27、0.78±0.17、2.01±0.6),模型组小鼠结肠中MDA含量和MPO酶活力显著增加(分别为2.2±0.48、1.6±0.22),T-AOC总抗氧化能力显著下降(0.88±0.28)。BSE组(MDA、SOD、GSH分别为1.67±0.51、1.02±0.36、1.44±0.47)和CCFM1252组(MDA、SOD、GSH分别为1.79±0.71、1.00±0.38、1.51±0.63)小鼠结肠的氧化应激水平均有一定程度的缓解,但BSE+CCFM1252组小鼠结肠MDA含量和MPO酶活力显著下降(分别为1.62±0.37、0.80±0.23),T-AOC总抗氧化能力显著增加(1.61±0.67),更接近空白组小鼠。说明鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物在缓解DSS引起的氧化应激方面效果更佳,且效果优于单独鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食。
实施例7:鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物可降低UC小鼠结肠促炎因子含量和表达水平
C57BL/6J小鼠分组、造模及处理方法同实施例4。
在第22天牺牲小鼠,收集小鼠结肠组织。一部分小鼠结肠组织按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,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。
另一部分用于测定小鼠结肠中TNFα、IL 1β、IL 6的转录水平。通过提取小鼠结肠的RNA,并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:5μL SYBR 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 time PCR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。引物序列如表2所示。
表2引物序列
实验结果如图6、图7所示,模型组小鼠结肠促炎因子TNF-α含量由空白组的47.15±6.6提高至66.53±6.12,BSE、CCFM1252、BSE+CCFM1252可分别降低至55.42±9.76、57.78±6.46、47.45±8.04。相比于空白组(IL-1β、IL-6分别为3.27±0.42、5.36±0.71),模型组小鼠结肠IL-1β含量(5.53±0.43)、IL-6含量(10.63±1.37)显著增加。而BSE组(4.38±0.79、6.54±0.61)、CCFM1252组(5.57±0.75、8.58±1.1)、BSE+CCFM1252组(3.91±0.76、6.1±1.35)均降低促炎因子的含量,其中IL-1β含量分别降低至4.38±0.79、5.57±0.75、3.91±0.76,IL-6含量分别降低至6.54±0.61、8.58±1.1、6.1±1.35。
促炎因子的表达水平结果显示,模型组小鼠结肠TNFα、IL 1β和IL 6表达水平分别为空白组的3.6倍、4.3倍、3.0倍。而灌胃含萝卜硫苷膳食后小鼠结肠TNFα、IL 1β和IL 6表达水平较模型组分别下降了60%、65%、65%;灌胃鼠李糖乳杆菌CCFM1252后小鼠结肠TNFα、IL 1β和IL 6表达水平较模型组分别下降了50%、20%、60%;灌胃鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物小鼠结肠TNFα、IL 1β和IL 6表达水平较模型组分别下降了77%、72%、66%。
以上结果表明,灌胃含萝卜硫苷膳食和鼠李糖乳杆菌CCFM1252均一定程度上减少了小鼠结肠促炎因子的释放,而鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物效果最佳。说明鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物能够显著缓解UC小鼠结肠炎症。
实施例8:鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物降低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)、鼠李糖乳杆菌CCFM1252组(CCFM1252)以及鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物组(BSE+CCFM1252)。
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,CCFM1252组灌胃含有5×109cfu/mL的菌悬液,BSE+CCFM1252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的脂多糖(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(0.25±0.02)显著高于空白组(0.34±0.01),说明模型组小鼠脾脏因LPS造成的肿大较为明显,而灌胃鼠李糖乳杆菌CCFM1252(0.32±0.02)、含萝卜硫苷膳食(0.32±0.02)以及鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物(0.31±0.1)均能显著缓解LPS引起的脾脏肿大。
实施例9:鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物降低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、IL6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,相比于空白组(TNFα、IL 1β、IL 6分别为29.04±4.85、3.51±075、4.89±0.71),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、9.98±0.97、10.20±1.92,而灌胃BSE、鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物的小鼠血清中促炎因子TNFα含量可分别下降至97.04±10.51、54±12.15和82.98±12.72,可使促炎因子IL-1β含量分别下降至9.34±1.44、8.45±1.59和6.02±1.16,使促炎因子IL-6含量分别下降至7.52±1.18、8.59±1.49和6.52±1.37。说明鼠李糖乳杆菌CCFM1252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10:鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物可降低LPS小鼠肝脏AST/ALT比率
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比例加入入预冷PBS缓冲液进行组织研磨,12000g,离心15min,取上清,分别用AST和ALT试剂盒测定小鼠肝脏中AST和ALT酶活力,并计算AST/ALT比值。
实验结果如图10所示,AST/ALT是肝功能指标,LPS引起的急性肝炎的情况下AST/ALT比值小于1。模型组小鼠肝脏AST/ALT比率(0.75±0.16)最高,AST/ALT比值越大说明肝脏损伤越严重;灌胃含萝卜硫苷膳食(0.70±0.06)对改善LPS引起AST/ALT比率增加效果不明显;而灌胃鼠李糖乳杆菌CCFM1252(0.60±0.14)和鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物(0.52±0.10)可显著降低AST/ALT,且鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物效果更好。说明鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物能够有效降低ASL/ALT比率,缓解LPS引起的肝损伤。
实施例11:鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物可降低LPS小鼠肝脏氧化应激水平
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比例加入入预冷PBS缓冲液进行组织研磨,12000g,离心15min,取上清,分别用MDA、SOD以及GSH-Px试剂盒测定小鼠肝脏中MDA含量、SOD和GSH-Px酶活力。
实验结果如图11所示,模型组小鼠肝脏中MDA含量(0.80±0.09)较空白组(0.58±0.05、76.68±2.85、3375.44±528.70)显著增加,SOD酶活力(69.82±2.81)和GSH-Px酶活力(2450±263.53)显著下降,说明LPS引起小鼠肝脏氧化应激水平提高。BSE组、CCFM1252组以及BSE+CCFM1252组小鼠肝脏中MDA含量(0.70±0.09、0.70±0.08、0.64±0.09)显著下降,SOD酶活力(81.29±3.65、73.13±4.41、83.11±3.25)和GSH-Px酶活力(3578.02±375.45、3202.51±552.25、3766.58±356.38)显著增加,且BSE+CCFMM1252组效果最好。说明鼠李糖乳杆菌和含萝卜硫苷膳食组合物能够显著降低LPS小鼠肝脏的氧化应激水平,具有良好的抗氧化作用,且效果优于单独灌胃鼠李糖乳杆菌CCFM1252或含萝卜硫苷膳食。
实施例12:鼠李糖乳杆菌CCFM1252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(BSE)组、鼠李糖乳杆菌CCFM1252和含萝卜硫苷膳食组合物(BSE+CCFM1252)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鼠李糖乳杆菌CCFM1252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的鼠李糖乳杆菌CCFM1252菌悬液。BSE组灌胃40mg/mL的西兰花种子水提物溶液,BSE+CCFM1252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。在第15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
实验结果如表5所示,BSE组和含萝卜硫苷膳食饲料组中均含有萝卜硫苷,因此经过肠道细菌的转化能在小鼠结肠内容物中检测到萝卜硫素,而正常饮食组无萝卜硫苷摄入,所以小鼠结肠内容物中无萝卜硫素。BSE+CCFM1252组和复合组小鼠内容物中萝卜硫素含量均高于BSE组和含萝卜硫苷膳食饲料组,说明鼠李糖乳杆菌CCFM1252可促进人体代谢萝卜硫苷生成萝卜硫素。
表5不同膳食处理小鼠粪便中萝卜硫素含量
实施例13:鼠李糖杆菌CCFM1252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、K2PO4·3H2O 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。
将鼠李糖乳杆菌CCFM1252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后冷冻干燥制得鼠李糖乳杆菌CCFM1252菌剂。
可选地,将所制菌剂与西兰花种子水提物混合,保证组合物中鼠李糖乳杆菌CCFM1252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,还可以利用鼠李糖乳杆菌CCFM1252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 (13)
1.一株能够生物转化萝卜硫素的鼠李糖乳杆菌(Lacticaseibacillus rhamnosus)CCFM1252,已于2022年3月6日保存至广东省微生物菌种保藏中心,保藏编号为 GDMCC NO:62273。
2.含有权利要求1所述鼠李糖乳杆菌CCFM1252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,所述益生菌制剂中鼠李糖乳杆菌CCFM1252的含量≥1×106CFU/g或1×106CFU/mL。
4.根据权利要求2或3所述的益生菌制剂,其特征在于,还含有西兰花种子水提物。
5.含有权利要求1所述鼠李糖乳杆菌CCFM1252的食品。
6.根据权利要求5所述的食品,其特征在于,还含有≥ 200 mg/g西兰花种子水提物。
7.含有权利要求1所述鼠李糖乳杆菌CCFM1252的药物。
8.根据权利要求7所述的药物,其特征在于,还含有≥ 200 mg/g西兰花种子水提物。
9.含有权利要求1所述鼠李糖乳杆菌CCFM1252的保健品。
10.根据权利要求9所述的保健品,其特征在于,还含有≥ 200 mg/g西兰花种子水提物。
11.权利要求1所述的鼠李糖乳杆菌CCFM1252在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。
12.权利要求1所述的鼠李糖乳杆菌CCFM1252在制备发酵食品中的应用。
13.权利要求1所述的鼠李糖乳杆菌CCFM1252在制备保健品中的应用。
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