CN111743159B - 复合微生物制剂及缓解抑郁和便秘的应用 - Google Patents
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Abstract
本发明公开了复合微生物制剂及缓解抑郁和便秘的应用,属于微生物技术领域。本发明制备的复合益生菌能够降低小鼠血清中促肾上腺释放激素和皮质酮的浓度,减轻由慢性压力导致的抑郁、焦虑样行为;所述复合益生菌能够上调肠道Htr4基因的表达,从而提高小鼠肠道蠕动能力;还能够提高肠道短链脂肪酸含量,对便秘具有显著改善作用;能够提高肠道紧密连接蛋白ZO‑1、Occludin、Claudin‑1的基因表达,降低肠道通透性,减少外周和中枢神经系统炎症;能够提高肠道、血清和大脑中5‑HTP的水平,具有改善睡眠的潜力。本发明制备的复合益生菌在抗抑郁和焦虑、缓解便秘、抗炎症、改善睡眠等方面具有广阔的应用价值。
Description
技术领域
本发明涉及复合微生物制剂及缓解抑郁和便秘的应用,具体涉及短双歧杆菌CCFM1025、长双歧杆菌婴儿亚种CCFM687和乳酸片球菌CCFM6432在制备抗抑郁和焦虑、缓解便秘、抗炎症、改善睡眠的药品和功能性食品中的应用,属于微生物技术领域。
背景技术
抑郁症是一类慢性且易复发的“精神感冒”,抑郁的产生可能与离别、环境压力、童年创伤、疾病以及基因等诸多因素有关。其常见的临床症状为情绪持续低落、社会孤立、疲惫失眠、产生自残自杀等消极观念。在临床治疗中,抑郁患者通常会伴随各类胃肠道疾病,其中便秘就是占比巨大的伴随症状。抑郁患者出现便秘症状可能与精神和心理状态密切相关,因为抑郁患者长期经受各种应激可能导致排便紊乱,进而诱发便秘;此外,长期使用的精神类药物可能诱发便秘,尤其是三环类抗抑郁药物具有明显的抗胆碱效应,可以拮抗副交感神经,导致胃肠动力不足,进而引发便秘。
目前,常规抗抑郁药物的开发主要是基于提高突触间隙具有改善情绪、提高动力的单胺神经递质。五羟色胺重摄取抑制剂(SSRIs)作为临床一线抗抑郁药物,它可以抑制5-羟色胺 (5-HT)被重摄取的过程,从而增加脑部中交流的信使分子。尽管这类新型抗抑郁药物具有良好的抗抑郁疗效,但是这类药物存在2-4周的起效迟滞期,这可能增加患者出现自杀的风险;此外,这类药物对三分之一左右的患者并不具有效果;更重要的是这些药物可能导致患者出现便秘、恶心、胀气等副作用。当抑郁患者出现便秘症状时,大多会采取泻药治疗,长时间使用泻药会形成严重的药物依赖,还有可能对肠道屏障造成损伤,严重者甚至出现结肠黑死病。基于上述情况,挖掘新的抗抑郁、缓解便秘的方法具有极大的市场价值和社会意义。
人体肠道内有1000-1500种细菌,活菌数量多达1012-1014,被称为“人体第二大基因库”。患有抑郁、焦虑、便秘、炎症等疾病的患者通常会出现肠道菌群失衡,主要体现为乳酸杆菌、双歧杆菌、拟杆菌等有益微生物的比例下降,而好氧真菌及致病菌的比例上升。益生菌是一类对宿主有益的活性微生物,可以参与肠道菌群的调控。大量研究已经证实三类双歧杆菌(长双歧、短双歧以及婴儿双歧)和四类乳杆菌(发酵乳杆菌、植物乳杆菌、瑞士乳杆菌、鼠李糖乳杆菌)具有潜在的抗抑郁功效。乳酸杆菌R0052和长双歧杆菌R0175因为在临床实验中具有良好的抗抑郁效果,已经成为市场化的产品。因此益生菌有望通过调节肠道微生物来缓解神经类疾病和胃肠道疾病。
在现有已经公开的专利及文献中有少数关于益生菌缓解抑郁症或者便秘的报道。例如 CN110066753A公开了植物乳酸菌DP189可以预防神经元的凋亡,促进神经营养因子的释放,在缓解抑郁症方面具有显著的效果;CN108823125A公开了一种具有治疗睡眠障碍、改善抑郁焦虑状态的益生菌制剂配方,主要是将冷冻干燥的副干酪益生菌和植物乳杆菌进行复配。此外,在专利CN103908585A中采用益生菌发酵中药提取物,这种益生菌发酵组合物可以作为预防和治疗便秘的新方法;CN106834187A公开了一种两歧双歧杆菌可以促进胃肠动力相关的活性肽的分泌,通过加速肠道的蠕动来缩短食糜排出的时间,从而缓解便秘。但是,这些专利菌株仅仅具有单方面的缓解精神障碍或者缓解胃肠道疾病的作用,并不能同时缓解抑郁症和便秘。
因此,筛选出可以有效缓解抑郁和便秘的复合益生菌至关重要。这对于益生菌在缓解精神类疾病以及胃肠道疾病的研究可以提供重要的参考作用。与此同时,这对于开发更高保健价值的益生菌有重要的意义,对于利用膳食策略缓解抑郁症和胃肠道疾病开辟新的路径和解决方案。
发明内容
本发明的第一个目的是提供一种复合益生菌制剂,含有短双歧杆菌CCFM1025、长双歧杆菌婴儿亚种CCFM687和乳酸片球菌CCFM6432,所述短双歧杆菌CCFM1025的保藏编号为GDMCC No.60386;所述长双歧杆菌婴儿亚种CCFM687的保藏编号为GDMCC No:60387;所述乳酸片球菌CCFM6432的保藏编号为GDMCC No.60638。
在一种实施方式中,所述短双歧杆菌(Bifidobacterium breve)CCFM1025,分类命名为 Bifidobacterium breve,已于2018年6月11日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:60386,并已公开于公开号为CN108949640A的专利申请文件中。
在一种实施方式中,所述长双歧杆菌婴儿亚种(Bifidobacterium longumsubsp.infantis) CCFM687,分类命名为Bifidobacterium longum subsp.infantis,已于2018年6月11日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:60387,并已公开于公开号为 CN109055269A的专利申请文件中。
在一种实施方式中,所述乳酸片球菌(Pediococcus acidilactici)CCFM6432,分类命名为 Pediococcus acidilactici,已于2019年4月25日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC No:60638,并已公开于公开号为CN110079485A的专利申请文件中。
在一种实施方式中,所述复合益生菌具有如下特性:
(1)减轻小鼠的抑郁样、焦虑样行为;
(2)降低小鼠血清中促肾上腺皮质激素、皮质酮的水平;
(3)提高小鼠肠道Htr4基因表达,增加肠道蠕动,缓解便秘;
(4)提高小鼠肠道紧密连接蛋白Claudin-1、ZO-1、Occludin的基因表达,降低肠道通透性;
(5)降低小鼠血清中促炎因子TNF-α的浓度;
(6)降低大脑前额叶皮质中促炎因子TNF-Α和IL-1β的浓度
(7)提高宿主肠道短链脂肪酸水平,改善炎症性肠病、腹泻等发生的风险;提高肠道、血清和大脑中5-HTP的水平,具有改善睡眠的潜在功效。
本发明的第二个目的是提供一种益生菌制剂,所述益生菌制剂含有短双歧杆菌CCFM1025、长双歧杆菌婴儿亚种CCFM687和乳酸片球菌CCFM6432三株益生菌。
在一种实施方式中,所述益生菌制剂中短双歧杆菌CCFM1025、长双歧杆菌婴儿亚种 CCFM687和乳酸片球菌CCFM6432按照活菌数比为1:1:1复配。
在一种实施方式中,每种菌的浓度≥1×105CFU/mL。
在一种实施方式中,每种菌的浓度≥1×105CFU/g。
本发明的第三个目的是提供一种发酵食品,所述发酵食品为使用复合益生菌发酵生产制得,所述发酵食品包括固态食品、液态食品、半固态食品。
在一种实施方式中,所述发酵食品包括乳制品、豆制品、果蔬制品,所述乳制品包括牛奶、酸奶油、干酪;所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
在一种实施方式中,所述发酵食品是在发酵初始接入所述复合益生菌进行发酵后制得。
本发明的第四个目的是提供复合益生菌在制备体内定植益生菌中的应用。
在一种实施方式中,所述应用是将三种菌株分别在MRS液体培养基中,37℃培养48小时,获得细胞培养液,以活菌数1:1:1的比例混合。
在一种实施方式中,用所述细胞培养液制备益生菌产品。
本发明的第五个目的是提供复合益生菌在制备抗抑郁、焦虑、缓解便秘、抗炎症、改善炎症性肠病、腹泻、睡眠的产品中的应用。
在一种实施方式中,所述产品为药物组合物。
在一种实施方式中,所述药物组合物还含有药学上可接受的载体。
本发明的第六个目的是提供所述的发酵食品在制备抗抑郁、焦虑、缓解便秘、抗炎症、改善炎症性肠病、腹泻、睡眠的功能性食品中的应用。
在一种实施方式中,所述复合益生菌能够降低小鼠血清中促肾上腺释放激素和皮质酮的浓度,缓解应激造成的下丘脑-垂体-肾上腺(HPA)轴亢奋,进而减轻由慢性压力导致的抑郁、焦虑样行为;相比于单菌,复合益生菌比单株菌的抗抑郁效果更好;所述复合益生菌能够上调肠道Htr4基因的表达,从而提高小鼠肠道蠕动能力;此外,还能够提高肠道短链脂肪酸,尤其是乙酸、丁酸的含量,对便秘具有显著改善作用;所述复合益生菌能够提高肠道紧密连接蛋白ZO-1、Occludin、Claudin-1的基因表达,降低肠道通透性,减少外周和中枢神经系统炎症;所述复合益生菌能够提高肠道、血清和大脑中5-HTP的水平,具有改善睡眠的潜力。因此,上述发酵食品可以有效降低抑郁、焦虑、便秘、炎症以及失眠等疾病发生的风险。
本发明的有益效果:在抑郁模型小鼠实验中,服用本发明的复合益生菌制剂能够降低小鼠血清中促肾上腺释放激素和皮质酮的浓度,缓解应激造成的下丘脑-垂体-肾上腺(HPA) 轴亢奋,进而减轻由慢性压力导致的抑郁、焦虑样行为;相比于单菌,复合益生菌比单株菌的抗抑郁效果更好;所述复合益生菌能够上调肠道Htr4基因的表达,从而提高小鼠肠道蠕动能力;此外,还能够提高肠道短链脂肪酸,尤其是乙酸、丁酸的含量,对便秘具有显著改善作用;所述复合益生菌能够提高肠道紧密连接蛋白ZO-1、Occludin、Claudin-1的基因表达,降低肠道通透性,减少外周和中枢神经系统炎症;所述复合益生菌能够提高肠道、血清和大脑中5-HTP的水平,具有改善睡眠的潜力。
本发明所述的复合益生菌能够用于制备具有抗抑郁、焦虑、缓解便秘、抗炎症、改善炎症性肠病、腹泻、睡眠效果的功能性食品、保健品和药品,具有非常广泛的应用前景。
生物材料保藏
短双歧杆菌(Bifidobacterium breve)CCFM1025,分类命名为Bifidobacteriumbreve,已于 2018年6月11日保藏于广东省微生物菌种保藏中心,保藏单位地址为:中国广州,保藏编号为GDMCC No:60386,并已公开于公开号为CN108949640A的专利申请文件中。
长双歧杆菌婴儿亚种(Bifidobacterium longum subsp.infantis)CCFM687,分类命名为 Bifidobacterium longum subsp.infantis,已于2018年6月11日保藏于广东省微生物菌种保藏中心,保藏单位地址为:中国广州,保藏编号为GDMCC No:60387,并已公开于公开号为CN109055269A的专利申请文件中。
乳酸片球菌(Pediococcus acidilactici)CCFM6432,分类命名为Pediococcusacidilactici,已于2019年4月25日保藏于广东省微生物菌种保藏中心,保藏单位地址为:中国广州,保藏编号为GDMCC No:60638,并已公开于公开号为CN110079485A的专利申请文件中。
附图说明
图1动物实验分组及处理方法;
图2为复合益生菌干预4周后的行为学变化示意图:(A)糖水偏好实验:(B)强迫游泳实验;(C)高架十字迷宫实验;其中#P<0.05,##P<0.01(vs正常对照组);*P<0.05,**P<0.01, ***P<0.001,****P<0.0001(vs抑郁模型组);
图3为复合益生菌干预4周后,实验鼠HPA轴亢奋程度示意图:(A)血清促肾上腺皮质激素;(B)血清皮质酮;其中##P<0.01,###P<0.001(vs正常对照组);*P<0.05,**P<0.01,***P<0.001(vs抑郁模型组);
图4为复合益生菌干预4周后实验鼠便秘相关指标变化示意图:(A)首粒黑便实验;(B) 小肠推进率实验;(C)结肠Htr4基因表达(vs模型组);其中##P<0.01,###P<0.001(vs正常对照组);*P<0.05,**P<0.01(vs抑郁模型组);
图5为复合益生菌干预4周后,实验鼠肠道通透性及血液炎症因子水平示意图:(A)结肠ZO-1基因表达量:(B)结肠Occludin基因表达量;(C)结肠Claudin-1基因表达量;(D)血清LPS;(E)血清TNF-α;其中##P<0.01,###P<0.001,####P<0.0001(vs正常对照组); *P<0.05,**P<0.01,***P<0.001,****P<0.0001(vs抑郁模型组);
图6为复合益生菌干预4周后,实验鼠前额叶皮质炎症因子水平示意图:(A)前额叶TNF-α;(B)前额叶IL-1β;其中#P<0.05,###P<0.001(vs正常对照组);**P<0.01,***P<0.001(vs抑郁模型组);
图7为复合益生菌干预4周后,实验鼠肠道、血清和前额叶皮质中5-HTP的含量示意图: (A)结肠5-HTP;(B)血清5-HTP;(C)前额叶5-HTP;其中#P<0.05,##P<0.01(vs正常对照组);*P<0.05,**P<0.01(vs抑郁模型组)。
具体实施方式
实施例1益生菌的分离筛选和鉴定
(一)益生菌的分离筛选:
(1)将从不同地区收集的粪便样品(粪便样品保存在30%的甘油中)吸取1mL,经过梯度稀释后,将粪便样品涂布在MRS固体培养基上,在厌氧工作站培养48h,厌氧工作站的温度为37℃;
(2)挑取菌落形态存在差异的菌株在MRS固体培养基上进行划线分离纯化,并且详细记录菌体的形态特征(表1);
(3)挑取培养基中分离出来的单菌,转移至MRS液体培养基,在37℃的厌氧工作站中培养48h,所得菌株进行革兰氏染色,记录菌落形态;
(4)弃除菌落中的革兰氏阴性菌菌株和革兰氏阳性球菌,挑选得到革兰氏阳性杆菌;
(5)过氧化氢酶分析后,弃除过氧化氢酶阳性菌株,保留过氧化氢酶阴性菌株。
表1三株益生菌的生理特性
(二)益生菌的分子生物学鉴定:
(l)益生菌的基因组抽提:
将获得的三株益生菌分别在MRS液体培养基中培养20h,所获得的益生菌菌液吸取1mL 于2mLEP管中,在1000rpm的条件下离心3min,弃上清液得到菌体;加入1mL无菌水,吹洗菌体后,在1000rpm条件下离心3min,所得沉淀即为菌体;加入200uLSDS裂解液,在 80℃30min;然后向菌体裂解液中加入酚-氯仿混合溶液,其成分及体积比为Tris饱和酚:氯仿:异戊醇=25:24:1,然后颠倒混匀,在12000rpm的条件下离心5min,取上清200uL;然后加入400uL的冰乙醇或冰异丙醇于200uL上清中,-20℃静置1h,然后在12000rpm的条件下离心5min,弃上清;加入500uL70%积分数)冰乙醇重悬沉淀,在12000rpm的条件下离心3min,弃上清;然后置于60℃烘箱烘干或者自然晾干;然后用50uLddH2O重新溶解沉淀得到模板DNA以进行PCR反应。
(2)16S rDNA PCR
A.PCR反应体系为50uL:10×Taq buffer,5uL;27F,0.5uL;1492R,0.5uL;Taq酶,0.5uL;模板,0.5uL;dNTP,5uL;ddH2O,38uL。
B.PCR反应条件为:95℃5min;95℃10s;55℃30s;72℃30s;step2-4 30×;72℃5min; 12℃2min。
C.结束后制备1%浓度的琼脂糖凝胶,将PCR产物与10000×loading buffer混合,上样量为2uL,在120V的电压下运行30min,然后进行凝胶成像分析。
D.结束后将PCR产物送至测序公司测定,将获得的测序序列在NCBI数据库中进行比对,初步鉴定得到三株菌为短双歧杆菌、长双歧杆菌婴儿亚种和乳酸片球菌。
(3)全基因组测序
将提取的全基因组送专业测序公司,利用二代测序仪对菌的全基因组进行测序,将得到的序列结果使用BLAST在GenBank中进行搜索和相似性比对,测序结果鉴定CCFM1025、 CCFM687、CCFM6432分别属于短双歧杆菌、长双歧杆菌婴儿亚种、乳酸片球菌中的新发现菌株。三株菌保藏在-80℃备用。
实施例2:复合益生菌能够缓解抑郁、焦虑症状
36只性C57BL/6J小鼠,5周龄,体重18-20g,SPF级。实验鼠适应环境一周后,根据体重随机分为三组:正常对照组、抑郁模型组、单菌干预组、复合益生菌干预组,每组含6只小鼠。实验鼠的生长环境条件为:环境温度(23±2)℃、相对湿度(50±10)%、光照模式(12h黑暗/12h光照)。实验鼠分组及饲养方案见图1。
采用慢性不可预知温和刺激造模,具体刺激因素包括:(1)禁食24h;(2)禁水24h;(3) 夹尾3min,夹子夹住尾尖1cm;(4)束缚2h;(5)强迫游泳10min,水温(21±2)℃;(6) 潮湿垫料24h;(7)空垫料24h;(8)持续光照24h;(9)孤养24h;(10)45度倾斜笼盒24h。每天采用1-2种不同的刺激,每次刺激的时间具有随机性,避免昼夜节律,在持续造模的4 周内,每种方法使用不超过三次。
复合益生菌制剂的制备:复合益生菌(短双歧杆菌CCFM1025、长双歧杆菌婴儿亚种CCFM687、乳酸片球菌CCFM6432)均来自于江南大学食品学院生物技术中心的菌种保存库。采用MRS液体培养基分别培养3株益生菌,厌氧工作站的环境温度为37℃,每隔20h进行一次传代,完成3次传代活化之后,按照2%的接种量进行扩配。所获得的菌液在8000g,4℃的条件下离心15min,采用30%甘油保存菌泥,在灌胃时使用10%浓度的脱脂乳溶解菌体,使复合益生菌的浓度到达109CFU/mL以上,实验鼠的灌胃剂量为0.4mL/只/天。
从第六周开始,停止慢性不可预知温和刺激和益生菌干预;同时开展糖水偏好、强迫游泳、高架十字迷宫三项行为学实验;结束后对抑郁相关理化指标进行测定。具体实施方法和结果如下:
(1)糖水偏好实验
先对实验鼠进行24h的糖水适应,在笼架上同时放置两个相同的水瓶,其中一个瓶盛装纯水,一个瓶盛装1%浓度的蔗糖水;适应结束后,实验鼠禁食禁水24h。实验开始前在每个饲养笼上放置一瓶纯水和一瓶1%浓度的蔗糖水,每隔3h记录一次糖水和纯水的消耗量,为了避免实验鼠饮水习惯带来的干扰,每隔1h,对调一次水瓶的位置。糖水偏好程度=糖水消耗量/(糖水消耗量+纯水消耗量)×100%。实验结果如图2A所示,抑郁模型组对糖水的偏好程度显著降低,而在补充复合益生菌之后,模型组对糖水偏好的程度得到明显的提升,这表明上述复合益生菌能够缓解慢性不可预知温和刺激导致的快感缺失。复配菌干预的小鼠的糖水偏好性(81.52%)显著高于三株单菌组(CCFM687:54.36%,P=0.002;CCFM1025:62.58%, P=0.026;CCFM6432:55.34%,P=0.003)。
(2)强迫游泳实验
强迫游泳实验是一种测定小鼠行为绝望的实验模型,在实验开始前24h让实验鼠适应性游泳5min,实验正式开始时,将实验鼠放入盛有水的5L烧杯中,水深10cm,水温(23±2℃),摄像机记录6min内小鼠的运动情况。分析实验鼠静止时间的占比,静止的标准为实验鼠放弃挣扎,漂浮于水面不动或者仅有轻微浮动。从图2B可以看出,当实验鼠置身于无可回避的压迫水域环境中,抑郁模型鼠的静止时间(59.92%)显著高于正常对照组(39.42%,P<0.001),这说明在面对应激时抑郁模型鼠更易放弃挣扎,更早进入行为绝望状态。在采用复合益生菌干预一个月后,抑郁实验鼠的静止时间显著缩短,抑郁症状得到有效缓解。
(3)高架十字迷宫实验
由于抑郁和焦虑的发病机理相似,且两种疾病有时会同时存在,为此开展高架十字迷宫实验评估实验鼠的焦虑状态。正式实验开始前,实验鼠提前30min放入行为学实验室适应环境。将实验鼠面向开臂放入十字架的中心区域,摄像机记录实验鼠在6min内的运动轨迹,为防止实验鼠在十字迷宫中残留的气味对实验造成干扰,相邻两次实验间隙,需要采用75%的酒精擦拭十字迷宫的四壁。分析实验鼠在开臂中停留的时间。高架十字迷宫可以通过实验鼠的自发探索行为来反应其焦虑程度,从图2C可以发现,与正常小鼠相比(21.14%),抑郁实验鼠在开臂中停留的时间显著降低(9.25%,P=0.016),某些小鼠几乎不进入开臂;相比于 CCFM687(29.61%)、CCFM1025(16.84%)和CCFM6432(23.67%),补充复合益生菌能大幅提高小鼠在开放臂停留时间比(50.81%)这说明复合益生菌能够增加实验鼠的探索行为,降低实验鼠的焦虑状态。单菌仅CCFM687和CCFM6432表现出显著的抗焦虑效果(P值分别为0.003和0.029),复配菌的效果大大优于三株单菌(P<0.001)。
(4)血清中促肾上腺皮质激素和皮质酮的测定
抑郁、焦虑的发生与HPA轴紊乱密切相关,研究表明过量的糖皮质激素进入脑部会导致神经元凋亡,并且扰乱神经内分泌系统,最终造成海马、前额叶等脑区的结构和功能受损;因此我们测定了HPA轴的关键调控分子促肾上腺皮质激素(ACTH)和皮质酮(CORT)的水平。实施例1中的实验鼠在第六周末进行眼球取血处死,获得的血浆静置2h,在3500rpm 的条件下离心15min,收集上层的血清。ACTH和CORT的测定采用酶联免疫吸附试剂盒(森贝伽,南京)。实验结果如图3所示,慢性不可预知温和刺激可以导致垂体过度分泌ACTH,过量的ACTH经过血液循环作用于肾上腺,导致肾上腺过度合成CORT,这也使得血清中ACTH和CORT的含量显著升高。摄入复合益生菌后,可以显著抑制ACTH和CORT的释放,从而缓解HPA轴的过度应答,显示出良好的抗抑郁、抗焦虑功效。相比于单菌,复配菌对ACTH和CORT过度应答的缓解效果更优。
实施例3:复合益生菌能够缓解慢性压力引起的便秘症状
(1)首粒黑便实验
首粒黑便实验可以用于模拟食糜在整个肠道的运动时间。在开始实验前,先对实验鼠禁食12h,然后灌胃0.2mL的活性炭阿拉伯树胶水溶液,将每只小鼠独立放置在装有吸水纸的收纳盒中;记录灌胃活性炭结束的时刻以及第一粒黑便产生的时刻,从而计算排出首粒黑便的总时间。结果如图4A所示,抑郁模型组和正常对照组排出首粒黑便的时间分别为257.5min、 167.8min,慢性应激导致粪便排出时间明显滞后,具有显著的便秘表观现象,而补充复合益生菌之后,可以使短粪便排出的时间缩短77.8min。
(2)小肠推进率实验
小肠推进率可以反应实验鼠小肠的运动能力。实验开始前,先对实验鼠进行12h禁食处理,然后灌胃0.2mL的活性炭阿拉伯树胶水溶液,30min后小鼠立即经眼球取血处死,然后打开胸腔,取下小鼠的胃到盲肠端的组织,记录小肠的总长度(幽门到盲肠的距离),测量墨汁从幽门沿着盲肠方向推进的长度。小肠推进率(%)=墨汁推进长度/小肠总长度×100%。结果表明(图4B)抑郁模型组的肠道运动能力更弱,推进率仅为44%,这表明肠道对食物的推进速度缓慢,到达盲肠需要更长的时间,而在补充复合益生菌之后,抑郁小鼠的肠道推进率显著提高了24.21%,因此该复合益生菌缓解便秘的效果显著。
(3)结肠Htr4的基因表达
5-HT具有14种不同的受体,这些受体分别在调控胃肠动力、免疫反应、骨骼生长等多个方面起着重要作用,Htr4是结肠上皮细胞中特有的一种G蛋白偶联受体,可以调节结肠的分泌功能以及加速肠道的传输能力,研究表明便秘型IBS以及慢性便秘与其密切相关,因此对结肠Htr4的基因表达情况进行分析。实施例1中的实验鼠在眼球取血处死之后,打开胸腔,取出小鼠结肠段组织,加入Trizol试剂裂解结肠组织提取样本中的RNA,按照反转录试剂盒的操作说明,将获得的RNA逆转录为更稳定的模板DNA,采用荧光染料SYBR混合样本,总反应体系为10uL,在Bio-Rad CFX384PCR仪器上进行定量分析,每个样本重复三次,结果采用2-ΔΔCt表示,所使用的目的基因的序列来自于Primer Bank,引物由上海生工生物公司合成。详细的基因序列见下表:
表2 qPCR引物序列
结果如图4C所示,抑郁模型组的Htr4的基因表达水平显著低于健康对照组,这表明慢性应激易导致机体出现便秘症状,这也与图4A和4B的结果相对应;在摄入复合益生菌后可以显著提高Htr4的基因表达水平,这可能是因为复合益生菌能够促进肠道的色氨酸转化为色胺,进而激活Htr4受体,加速结肠的传输功能,最终能够逆转便秘症状。
(4)肠道短链脂肪酸的测定
短链脂肪酸(SCFAs)是肠道厌氧微生物在结肠内发酵碳水化合物所获得的一类饱和脂肪酸;其中乙酸、丙酸、丁酸是肠道中占比最多的三种SCFAs,采用结肠管腔注射的方式为豚鼠补充乙酸盐、丙酸盐和丁酸盐,结果发现乙酸盐和丁酸盐可以促进结肠运动,因此乙酸和丁酸可能具有缓解便秘的潜力。取实验鼠盲肠内容物(湿重>50mg)。盲肠内容物经过冷冻干燥去除水分,加入500uL饱和氯化钠溶液浸泡,使用均质机粉碎盲肠内容物之后,加入40uL10%浓度的硫酸进行酸化,然后加入1mL无水乙醚提取短链脂肪酸。所得的混合体系在14000g,4℃的条件下离心15min,将溶解有短链脂肪酸的上层有机相转移到盛装有0.25g无水硫酸钠的2mLEP管中,在上述条件下再次离心,吸取500uL的上清液于气相瓶中待用。采用GC-MS(TSQ900,Thermo Scientific)测定样品中的六种短链脂肪酸(乙酸、丙酸、丁酸、异丁酸、戊酸、异戊酸)的含量。选用的分离柱为Rtx-Wax毛细管柱;采用氦气作为载气,流速为0.89mL/min;采用程序升温,柱箱温度按照7.5℃/min的速率,从100℃上升到 140℃,然后以6℃/min的速率上升至200℃;离子源和界面的温度分别为220℃和250℃,质谱仪扫描范围m/Z为2-100。
表3复合益生菌干预4周后小鼠盲肠内容物中短链脂肪酸的含量(单位:pmol/mg)
注:*P<0.05,**P<0.01,***P<0.001,****P<0.0001(vs模型组)。
结果如表3所示,具有便秘症状的实验鼠与正常对照组之间的乙酸和丁酸含量具有非常显著的差异,这提示上述两种短链脂肪酸可能与便秘的产生相关。在服用复合益生菌后,抑郁实验鼠盲肠内容物中短链脂肪酸的总量增加,且每种短链脂肪酸呈现不同程度的增加,其中丁酸和乙酸的含量得到显著提高,并与空白对照的含量近似。现有的研究表明丁酸盐可以刺激肠道神经元分泌血清素和肠道动力相关的激素,而乙酸通过调节结肠调节型T细胞的体内平衡来维持肠道的运动性,通过加速结肠的蠕动,提高肠道的运动能力,因此可以促进排便,在缓解便秘症状方面具有潜在的价值。
实施例4:复合益生菌能够降低肠道通透性,减轻外周和中枢系统炎症反应
将按照实施例1的方法处理的实验鼠在眼球取血之后,迅速剥离小鼠的脑组织,并在冰块上分离出前额叶组织,取下一部分前额叶组织(>50mg),按照1:9(组织:裂解液)的比例加入裂解液(即1g组织,加入9g裂解液)。为了防止蛋白降解,裂解液中加入蛋白酶抑制剂、磷酸酶抑制剂和EDTA螯合剂。在均质机上破碎组织,获得的组织液在14000g,4℃的条件下离心15min,获得的上清液即为前额叶匀浆液,炎症因子TNF-α和IL-1β的测定采用试剂盒(R&D,美国),具体方法参考说明书进行。实验如图5所示,结果表明慢性不可预知温和刺激会导致免疫系统失衡,长期处于应激状态会使得前额叶中TNF-α和IL-1β两种炎症因子的水平分别升高了146.07%(P=0.027)和143.23%(P<0.001)(图6),引发外周和中枢神经系统产生炎症反应。而在采用复合益生菌干预之后,抑郁实验鼠的脑部炎症水平得到有效缓解,并且接近于正常水平(与对照组比较的P值分别为0.058和0.893,即无统计学差异),这说明复合益生菌在抵抗炎症、维持免疫平衡等方面具有潜在的价值。
在现有的研究中,免疫炎症反应与肠道通透性密切相关,为此对三种重要紧密连接蛋白 Claudin-1、ZO-1、Occludin的基因表达进行测定。结肠组织的处理方法参考实例2中Htr4基因表达的测定方法。引物由上海生工生物公司合成。详细的基因序列见下表:
表4 qPCR引物序列
结果如图5所示,相比于正常对照组,抑郁模型组的三种紧密连接蛋白Claudin-1、ZO-1、 Occludin的基因表达量显著降低,这提示应激可能通过影响上述三种紧密连接蛋白来损害肠道屏障。当机体的肠道通透性增加,肠道内的病原体以及炎症因子更易通过血液循环引发外周和中枢免疫应答,这也和先前发现的抑郁模型组血清和前额叶中TNF-α和IL-1β水平显著升高一致。而复合益生菌能够使实验鼠结肠内的Claudin-1、ZO-1、Occludin基因表达量显著提高,这意味着复合益生菌干预后的抑郁实验鼠的肠道通透性降低。因此复合益生菌在维持肠道屏障稳定和调控中枢系统炎症反应方面起着重要作用。
实施例5:复合益生菌能够提高大脑5-HTP的水平
5-HT是一种在机体分布广泛的生物性吲哚胺,可直接参与抑郁、焦虑、失眠等诸多疾病的调控。L-Trp是合成5-HT的前体物质,在色氨酸羟化酶(Tryptophan hydroxylase,TPH) 的作用下,转化为5-羟色氨酸(5-hydroxytryptophan,5-HTP),5-HTP在芳香族氨基酸脱羧酶的作用下进一步脱羧生成5-HT(Yano J M.et al.Cell,2015,161(2):264)。机体90%以上的 5-HT是由位于结肠的肠嗜铬细胞产生,但是由于血脑屏障的存在,外周的5-HT并不能进入中枢系统,但其前体物质5-HTP可以穿透血脑屏障,为此对结肠、血清以及前额叶中的5-HTP 进行测定。前额叶和结肠匀浆液的制备参考实施例3中的前额叶组织处理方法。取100uL匀浆液,加入100uL质量分数为5%的高氯酸沉淀蛋白,在12000g,4℃的条件下离心10min,取上清液,过0.22um玻璃滤器,储存于液相瓶中待用。流动相中的水相为0.1mol/L的乙酸钠溶液,有机相为纯甲醇,流动相比例为85:15(水相:有机相)。采用高效液相系统(Waters 2695)和配套的荧光检测系统(Waters 2475)分析测定5-HT/5-HTP(上述物质的标准品购买于Sigma-Aldrich公司),分析条件为发射波长330nm,激发波长290nm,分离的色谱柱为C18 柱(Inert Sustain C18,150mm×4.6mm,5um)。从图7中可以看出,在采用复合益生菌干预之后,上述三个区域中的5-HTP的浓度显著升高,这表明复合益生菌能够刺激肠道微生物合成5-HTP,并且通过血液循环到达中枢神经系统,为5-HT的合成提供充足的前体物质。
早期研究表明,给大鼠的中缝背核注射微量具有抑制5-HT合成作用的对氯苯丙氨酸可以制造典型的失眠模型(肖成荣等,毒理学杂质,2007,21(4):326);而在杏仁核通过注射 5-HT能够促进慢波睡眠和快波睡眠,可明显改善失眠(朱国庆等,中国药理学通报,1999, 15(2):142);此外,在临床中也有案例表明,口服5-HTP用于治疗失眠,可以使患者的失眠状态得到不同程度改善(Fischer PC.et al.Elect Clin Neuroph,1974,36(1):1-8);而5-HT重摄取抑制剂帕罗西汀也被美国食品和药物管理局批准成为治疗女性更年期热潮红和睡眠障碍的特效药(Rahimzadeh P.et al.Cancer Manag Res,2018,10:4831-4837)。由此可见5-HTP 和5-HT是调节睡眠与觉醒的重要神经递质,且复合益生菌对改善睡眠有一定的效果。
实施例6:利用本发明使用的复合益生菌制备发酵食品
选用新鲜蔬菜洗净后榨汁,接着进行高温瞬间灭菌,在温度140℃下高温热杀菌2秒后,立即降温至37℃,再接入本发明制备的复合益生菌发酵剂,使其浓度达到109CFU/mL以上,在温度4℃下冷藏保存,于是得到含有本发明复合益生菌的果蔬饮料。
利用本发明能够使用复合益生菌发酵生产制备其他发酵食品,所述发酵食品包括固态食品、液态食品、半固态食品。所述发酵食品包括乳制品、豆制品、果蔬制品,所述乳制品包括牛奶、酸奶油、干酪;所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
所述复合益生菌能够降低小鼠血清中促肾上腺释放激素和皮质酮的浓度,缓解应激造成的下丘脑-垂体-肾上腺(HPA)轴亢奋,进而减轻由慢性压力导致的抑郁、焦虑样行为;相比于单菌,复合益生菌比单株菌的抗抑郁效果更好;所述复合益生菌能够上调肠道Htr4基因的表达,从而提高小鼠肠道蠕动能力;此外,还能够提高肠道短链脂肪酸,尤其是乙酸、丁酸的含量,对便秘具有显著改善作用;所述复合益生菌能够提高肠道紧密连接蛋白ZO-1、 Occludin、Claudin-1的基因表达,降低肠道通透性,减少外周和中枢神经系统炎症;所述复合益生菌能够提高肠道、血清和大脑中5-HTP的水平,具有改善睡眠的潜力。因此,上述发酵食品可以有效降低抑郁、焦虑、便秘、炎症以及失眠等疾病发生的风险。
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。
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Claims (7)
1.一种复合益生菌制剂,其特征在于:活性成分为短双歧杆菌CCFM1025、长双歧杆菌婴儿亚种CCFM687和乳酸片球菌CCFM6432,所述益生菌制剂中短双歧杆菌CCFM1025、长双歧杆菌婴儿亚种CCFM687和乳酸片球菌CCFM6432按照活菌数比为(1~1.5):(1~1.5):(1~1.5)复配。
2.根据权利要求1所述的益生菌制剂,其特征在于,每种菌的浓度≥1×105CFU/mL或1×105CFU/g。
3.一种发酵食品,其特征在于,所述发酵食品使用权利要求1-2任一项所述的复合益生菌制剂发酵生产制得,所述发酵食品为固态食品、液态食品或半固态食品。
4.根据权利要求3所述的发酵食品,其特征在于,所述发酵食品为乳制品、豆制品或果蔬制品。
5.根据权利要求4所述的发酵食品,其特征在于,所述乳制品为牛奶、酸奶油或干酪;所述果蔬制品为黄瓜、胡萝卜、甜菜、芹菜或圆白菜制品。
6.根据权利要求3-5任一项所述的发酵食品,其特征在于,所述发酵食品是在发酵初始接入所述复合益生菌制剂进行发酵后制得。
7.权利要求1所述的复合益生菌制剂在制备缓解抑郁、缓解便秘以及抗炎症的药品中的应用。
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