CN115721017A - 一株可激活肠道抗炎靶点的短双歧杆菌及应用 - Google Patents
一株可激活肠道抗炎靶点的短双歧杆菌及应用 Download PDFInfo
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- CN115721017A CN115721017A CN202211406842.8A CN202211406842A CN115721017A CN 115721017 A CN115721017 A CN 115721017A CN 202211406842 A CN202211406842 A CN 202211406842A CN 115721017 A CN115721017 A CN 115721017A
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Abstract
本发明公开了一株可激活肠道抗炎靶点的短双歧杆菌及应用,属于微生物技术领域。本发明提供了短双歧杆菌CCFM683在改善肠道免疫方面的新用途。本发明利用短双歧杆菌CCFM683激活肠道抗炎靶点,显著提高了抗炎靶点PPAR‑γ和GPR40的表达量。该菌株可作为现有的PPAR‑γ的激动剂或GPR40激动剂的益生菌替代产品,具有广阔的市场前景。
Description
技术领域
本发明涉及一株可激活肠道抗炎靶点的短双歧杆菌及应用,尤其是一种可激活肠道抗炎靶点的菌株,其可添加在各种健康食品及保健食品中,属于微生物技术领域。
背景技术
肠道屏障是指能够阻止肠腔内病原菌和毒素穿过肠黏膜入侵到血液系统和其它组织器官的结构和功能的总和,对人体健康起重要作用。肠道屏障由机械屏障、免疫屏障、生物屏障和化学屏障共同构成。肠道黏液层、上皮细胞层及紧密连接构成了肠道的第一道防线-肠道机械屏障,起维护肠道屏障功能的作用;肠道化学屏障包括溶菌酶、胆汁酸、各种消化酶等,能阻止致病因子入侵;肠道菌群(细菌、真菌和病毒)构成了肠道生物屏障,能分泌细菌毒素抑制致病菌,维护肠道健康;肠道免疫屏障(淋巴组织、免疫细胞、炎症因子等)是肠道免疫的核心,在抵御肠道炎症中发挥重要的作用。
许多肠道相关的疾病,如结肠炎,易激性肠炎,腹泻,便秘等肠道疾病都与肠道免疫屏障的破坏直接相关。结肠炎,便秘,腹泻,易激性肠炎等肠道疾病会对肠道造成较大的影响,会影响患者的生活质量,导致社会、心理和专业领域的变化。使用药物进行常规治疗的研究越来越多,目的是减少症状和炎症。长期使用肠道肠道炎症相关的药物(抑制剂类,激素类,抗生素类)可能会导致高血压、糖尿病、骨质疏松等其他疾病,从而影响治疗的成功。
鉴于现有治疗方案存在的多种问题,替代传统方法的方案显得尤为重要了,新的治疗方案包括单克隆抗体、益生元、益生菌等。而上述多种疗法的可行性也促使我们继续寻找应用更广泛、潜力更巨大,同时又具有调节肠道免疫作用的膳食补充剂。益生菌对便秘、肠炎、腹泻、非酒精性脂肪肝、糖脂代谢紊乱、情绪及行为障碍均具有显著改善作用,而这与益生菌对肠道免疫的调节密切相关。因此,筛选对肠道免疫起作用的益生菌显得尤为重要。
PPAR-γ是PPARα、PPARβ/δ和PPAR-γ的三种亚型之一,其中PPAR-γ属于核受体超家族。PPAR-γ是肠道中一个重要的抗炎介质和靶点,研究表明PPAR-γ的激活可以减轻肠道的炎症反应。G蛋白偶联受体(G Protein-Coupled Receptors,GPCRs)是生理上重要的膜蛋白,能感知信号分子。G蛋白偶联受体40(GPR40),也被称为游离脂肪酸受体1(FFAR1),据报道可被一系列6个碳链的中长链饱和脂肪酸和不饱和脂肪酸激活,GPR40激活能调节肠道炎症。因此,PPAR-γ和GPR40是肠道重要的抗炎靶点。因此,筛选能激活肠道PPAR-γ和GPR40靶点的益生菌对改善肠道炎症显得尤为重要。
发明内容
本发明提供了短双歧杆菌(Bifidobacterium breve)CCFM683在制备改善肠道免疫的产品中的应用;所述短双歧杆菌CCFM683已于2015年12日04日保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心,保藏编号为CGMCC No.11828,并公开于公开号为CN106038611A。
在一种实施方式中,所述产品为药物或保健品。
在一种实施方式中,所述产品为预防肠道炎症的药物。
在一种实施方式中,所述药物具有激活抗炎靶点PPAR-γ和/或激活抗炎靶点GPR40的功能。
在一种实施方式中,所述短双歧杆菌CCFM683在药物中的含量≥1×108CFU/g或1×108CFU/mL。
在一种实施方式中,所述药物还含有药学上可接受的辅料。
在一种实施方式中,所述药学上可接受的辅料包括填充剂、粘合剂、润湿剂、崩解剂、润滑剂、矫味剂中的一种或多种。
在一种实施方式中,所述药物的剂型为颗粒剂、胶囊剂、片剂、丸剂或口服液。
在一种实施方式中,所述药物含有所述短双歧杆菌CCFM683的湿细胞或冻干后的细胞。
在一种实施方式中,所述保健品是短双歧杆菌CCFM683的微生物制剂。
在一种实施方式中,所述微生物制剂中短双歧杆菌CCFM683的活菌数≥1×106CFU/g。
在一种实施方式中,所述微生物制剂的制备方法为:将短双歧杆菌CCFM683接种到mMRS培养基中,37℃厌氧条件下培养18-20h,收集菌体,用保护剂重悬菌体细胞,使菌浓度达到1×1010CFU/mL,然后悬浮液在37℃厌氧条件下培养50-70min,干燥。
在一种实施方式中,每升所述mMRS培养基含有:胰蛋白胨10g、牛肉浸膏10g、酵母粉5g、葡萄糖20g、柠檬酸氢二铵2g、醋酸钠5g、磷酸氢二钾2g、七水硫酸镁0.5g、一水合硫酸锰0.25g,吐温80 1mL与0.5g半胱氨酸。
在一种实施方式中,所述保护剂含有脱脂奶粉、麦芽糊精、海藻糖。
在一种实施方式中,所述保护剂含有:100g/L-150g/L脱脂奶粉、100g/L-150g/L麦芽糊精、140g/L-160g/L海藻糖。
在一种实施方式中,将收集的菌体用磷酸盐缓冲液清洗2-4次,所述磷酸盐缓冲液pH为6.8-7.2。
在一种实施方式中,所述干燥可以采用任意一种菌液干燥工艺进行干燥,包括但不限于真空冷冻干燥。
在一种实施方式中,所述真空冷冻干燥是在-15~-20℃预冻8-14h后进行真空冷冻干燥。
本发明还提供所述短双歧杆菌CCFM683在制备PPAR-γ激动剂或GPR40激动剂中的应用。
有益效果:
(1)本发明所提供的短双歧杆菌(Bifidobacterium breve)CCFM683分离自新生儿的肠道菌群,该菌株对人体无毒副作用,因此采用本发明提供的短双歧杆菌CCFM683制备的药物,相对于用于调节肠道炎症的传统药物具有一定的优势,并且该菌株可用于制作益生菌制剂等,具有广阔的市场前景;
(2)本发明提供了短双歧杆菌CCFM683在激活抗炎靶点PPAR-γ和激活抗炎靶点GPR40方面的新用途,可用于制备改善肠道免疫的保健食品或药物,具有非常广泛的应用前景。
附图说明
图1:短双歧杆菌CCFM683对小鼠结肠组织中PPAR-γ的影响;
图2:短双歧杆菌CCFM683对小鼠结肠组织中GPR40的影响。
图1~2中,“*”、“**”、“***”、“****”均表示与空白组具有显著性差异,*越多,显著性差异越大,误差以Mean±SEM的形式展现。
具体实施方式
下面结合具体实施例和附图对本发明进行进一步的阐述。
下述实施例中涉及的小鼠为8周龄雄性SPF(Specific pathogen free,无特定病原体)级C57BL/6J小鼠,购自集萃药康;下述实施例中涉及的脱脂乳粉、海藻糖、蔗糖、多聚甲醛购自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:
mMRS液体培养基:胰蛋白胨10g/L、牛肉膏10g/L、酵母粉5g/L、葡萄糖20g/L、无水乙酸钠2g/L、七水硫酸镁0.5g/L、一水硫酸锰0.25g/L、柠檬酸氢二铵2g/L、三水磷酸氢二钾2.6g/L、Tween80 1mL/L、半胱氨酸盐酸盐0.5g/L。
mMRS固体培养基:胰蛋白胨10g/L、牛肉膏10g/L、酵母粉5g/L、葡萄糖20g/L、无水乙酸钠2g/L、七水硫酸镁0.5g/L、一水硫酸锰0.25g/L、柠檬酸氢二铵2g/L、三水磷酸氢二钾2.6g/L、Tween80 1mL/L、半胱氨酸盐酸盐0.5g/L,琼脂20g/L。
下述实施例中涉及的检测方法如下:
蛋白质印迹实验:
蛋白样本的提取及浓度测量:将保存在-80℃冰箱中的结肠组织取出,在冰上剪碎,称取约50mg组织于装有5颗氧化锆珠的2mL离心管中,加入约500uL的RIPA裂解液(按量添加蛋白酶抑制剂和磷酸酶抑制剂);高通量组织破碎,然后离心(4℃,12000g)10min,收集上清,利用BCA试剂盒测量蛋白浓度,根据蛋白浓度,利用RIPA裂解液将所有样本的蛋白的浓度调整为2.5mg/mL。然后按4:1(v:v)加入5×Loading Buffer混匀后,沸水浴加热10min。-20℃保存备用。蛋白的制胶,电泳,转膜,封闭,一抗孵育,二抗孵育,成像等的具体方法参考朱升龙的博士论文(朱升龙[D].江南大学,2018.)。
实施例1:短双歧杆菌CCFM683菌悬液的制备
(1)从甘油管中蘸取短双歧杆菌CCFM683的菌液在mMRS固体培养基上划线,厌氧环境下37℃培养48h,得到单菌落;挑取单菌落接种于mMRS液体培养基中,厌氧环境下37℃培养48h进行活化培养,重复此操作3次,得到活化后的菌液。
(2)将步骤(1)得到的活化后的菌液按照按2%(v/v)的接种量接种至mMRS液体培养基中,37℃培养24h后得到发酵液,将发酵液离心收集菌体,用生理盐水重悬菌体,并调整活菌数5×109CFU/mL,制成菌悬液。
实施例2:短双歧杆菌CCFM683对小鼠结肠组织中PPAR-γ的影响
动物处理的步骤如下:
(1)取8周龄健康雄性C57BL/6J小鼠16只,随机分为2组,2组分别命名为:对照组(Control)、短双歧杆菌CCFM683干预组(CCFM683);采用每组8只,实验方案和各组小鼠的处理方式如表1所示。
(2)对对照组(Control)、短双歧杆菌CCFM683处理组(CCFM683)进行处理:
其中,短双歧杆菌CCFM683干预组的处理方法为:实验第1-14天,每天给短双歧杆菌CCFM683干预组灌胃5×109CFU/mL短双歧杆菌CCFM683菌悬液200μL,自由饮用蒸馏水。
对照组(Control)的处理方法为:实验第1-14天,每天给对照组灌胃生理盐水200μL,自由饮用蒸馏水。
表1实验小鼠处理方案
取结肠组织按照结肠组织生化指标测定方法检测结肠组织上清中PPAR-γ的浓度。PPAR-γ在结肠炎症反应过程中是一个重要的抗炎介质,PPAR-γ的靶点紊乱能够增加小鼠对肠道屏障损坏的敏感性。因此,对短双歧杆菌CCFM683作用的小鼠结肠组织中的PPAR-γ进行了研究。免疫印迹法显示,与空白组相比(0.65),短双歧杆菌CCFM683干预后显著提高了PPAR-γ的浓度,相对浓度达到0.91(图1)。因此,短双歧杆菌CCFM683是一种有效的PPAR-γ的激动剂。
实施例3:短双歧杆菌CCFM683对小鼠结肠靶点GPR40的影响
实验方法同实施例2中的步骤(1)~(2);取结肠组织按照结肠组织生化指标测定方法检测结肠组织上清中GPR40的浓度。G蛋白偶联受体40(GPR40)的靶点紊乱能够增加小鼠对肠道屏障损坏的敏感性。因此,对短双歧杆菌CCFM683作用的小鼠结肠组织中的GPR40进行了研究。免疫印迹法显示,与空白组相比(0.07),短双歧杆菌CCFM683干预后显著提高了GPR40的浓度,相对浓度达到0.25(图2)。因此,短双歧杆菌CCFM683是一种有效的GPR40的激动剂。
实施例4:制备含短双歧杆菌CCFM683的发酵剂
mMRS培养基:胰蛋白胨10g、牛肉浸膏10g、酵母粉5g、葡萄糖20g、柠檬酸氢二铵2g、醋酸钠5g、磷酸氢二钾2g、七水硫酸镁0.5g、一水合硫酸锰0.25g,吐温80 1mL,半胱氨酸0.5g,水定容至1000mL,调节pH至6.5,在119-123℃条件下灭菌15-25min。
保护剂:100g/L-150g/L脱脂奶粉、100g/L-150g/L麦芽糊精、140g/L-160g/L海藻糖。
将短双歧杆菌CCFM683接种到mMRS培养基中,37℃厌氧条件下培养18-20h,收集菌体,用保护剂重悬菌体细胞,使菌浓度达到1010CFU/mL,然后悬浮液在37℃厌氧条件下培养50-70min,干燥。
可选地,所述干燥是在-15~-20℃预冻8-14h后进行真空冷冻干燥。
实施例5:短双歧杆菌CCFM683的应用
(1)利用短双歧杆菌CCFM683制造乳饮料
将原料乳脱脂奶在95℃热杀菌20min,然后冷却至4℃,再加入短双歧杆菌CCFM683或实施例4的短双歧杆菌CCFM683发酵剂,使菌体浓度达到106CFU/mL以上,在4℃冷藏保存即得到含短双歧杆菌CCFM683活菌的乳饮料。
(2)利用短双歧杆菌CCFM683制造豆奶
采用软水浸泡大豆,水量为原大豆量三倍体积,在温度80℃下浸泡1~2h,再去除大豆皮。接着,沥去浸泡水,另加沸水磨浆,并在温度高于80℃的条件下保温10~15min。浆体用150目滤膜过滤后离心,得到的离心液即为粗豆奶,再将它加热到温度140~150℃,然后将热的粗豆奶迅速导入真空冷却室进行抽真空,所述粗豆奶中的异味物质随着水蒸汽迅速排出。经过真空脱气后,将其温度降至37℃左右,再接入短双歧杆菌CCFM683或实施例4制备的发酵剂,使短双歧杆菌CCFM683浓度达到106CFU/mL以上,在4℃冷藏保存即得到含短双歧杆菌CCFM683活菌的豆奶。
(3)利用短双歧杆菌CCFM683制造果蔬饮料
选用新鲜蔬菜(例如黄瓜、胡萝卜、甜菜、芹菜或圆白菜中的一种或多种)洗净后榨汁,接着进行高温瞬间灭菌,在温度140℃下高温热杀菌2s后,立即降温到37℃左右,再接入本发明的短双歧杆菌CCFM683发酵剂,使其浓度达到106CFU/mL以上,在4℃冷藏保存即得到含短双歧杆菌CCFM683活菌的果蔬饮料。
(4)利用短双歧杆菌CCFM683制胶囊制品
本发明的短双歧杆菌CCFM683在mMRS培养基上培养24h,在温度4℃与4000r/min的条件下离心20min,用PBS冲洗两次,再加入以最后得到含短双歧杆菌CCFM683的粉剂重量计4%脱脂奶粉和6%乳糖混合10min,再加入无菌2%氯化钙和3%海藻酸钠,同时以150r/min搅拌10min,再静止固化30min,最后清洗过滤,得到的滤液进行冷冻干燥20h,得到含短双歧杆菌CCFM683的粉剂,把这种粉剂装入商业化的药用微胶囊,得到所述的胶囊制品。
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 (10)
1.短双歧杆菌(Bifidobacterium breve)CCFM683在制备改善肠道免疫的产品中的应用,其特征在于,所述短双歧杆菌CCFM683保藏编号为CGMCC No.11828。
2.根据权利要求1所述的应用,其特征在于,所述产品为药物或保健品。
3.根据权利要求2所述的应用,其特征在于,所述产品为激活肠道抗炎靶点的药物。
4.根据权利要求3所述的应用,其特征在于,所述抗炎靶点包括但不限于PPAR-γ和/或GPR40。
5.根据权利要求2~4任一所述的应用,其特征在于,所述短双歧杆菌CCFM683在药物中的含量≥1×108CFU/g或1×108CFU/mL。
6.根据权利要求2所述的应用,其特征在于,所述产品是保护肠道免疫屏障的保健品。
7.根据权利要求6所述的应用,其特征在于,所述保健品是短双歧杆菌CCFM683的微生物制剂。
8.根据权利要求7所述的应用,其特征在于,所述微生物制剂中短双歧杆菌CCFM683的活菌数≥1×106CFU/g。
9.根据权利要求8所述的应用,其特征在于,所述微生物制剂的制备方法为:将短双歧杆菌CCFM683接种到mMRS培养基中,培养一段时间后收集菌体,用保护剂重悬菌体细胞,然后将菌体悬浮液培养一段时间后干燥。
10.短双歧杆菌CCFM683在制备PPAR-γ的激动剂或GPR40激动剂中的应用,其特征在于,所述短双歧杆菌CCFM683保藏编号为CGMCC No.11828。
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CN105925514A (zh) * | 2016-07-12 | 2016-09-07 | 江南大学 | 一株短双歧杆菌及其制备共轭亚油酸或共轭亚麻酸的应用 |
CN106038611A (zh) * | 2016-07-12 | 2016-10-26 | 江南大学 | 短双歧杆菌c11及其用途 |
CN113106052A (zh) * | 2021-05-31 | 2021-07-13 | 江南大学 | 一种提升短双歧杆菌增殖效率的肽及其应用 |
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CN106038611A (zh) * | 2016-07-12 | 2016-10-26 | 江南大学 | 短双歧杆菌c11及其用途 |
CN113106052A (zh) * | 2021-05-31 | 2021-07-13 | 江南大学 | 一种提升短双歧杆菌增殖效率的肽及其应用 |
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Title |
---|
孙庆申;罗钦文;许艳玲;: "双歧杆菌及其产品缓解肠炎症状的研究进展", 乳业科学与技术, no. 04, pages 29 - 33 * |
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