CN109628343B - 短双歧杆菌yh68及在降低鼠伤寒沙门氏菌感染风险产品中的应用 - Google Patents
短双歧杆菌yh68及在降低鼠伤寒沙门氏菌感染风险产品中的应用 Download PDFInfo
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- CN109628343B CN109628343B CN201811602269.1A CN201811602269A CN109628343B CN 109628343 B CN109628343 B CN 109628343B CN 201811602269 A CN201811602269 A CN 201811602269A CN 109628343 B CN109628343 B CN 109628343B
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Abstract
本发明提供了一株短双歧杆菌YH68及其在降低鼠伤寒沙门氏菌感染风险的产品中的应用,涉及益生菌的应用技术领域。本发明所述短双歧杆菌YH68的生物保藏编号为CGMCC NO.14096。本发明所述短双歧杆菌YH68可通过破坏鼠伤寒沙门氏菌细胞膜的完整性以及下调编码侵袭蛋白的invF基因表达量,下调编码入侵宿主细胞关键的转录调控基因hilA、编码I型菌毛的结构蛋白基因fimA、以及编码效应蛋白的基因sipA和sipB的表达量,从而抑制鼠伤寒沙门氏菌活性,可用于制备降低鼠伤寒沙门氏菌感染风险的食品、补充剂或药物。
Description
技术领域
本发明属于益生菌的应用技术领域,具体涉及短双歧杆菌YH68及其在降低鼠伤寒沙门氏菌感染风险产品中的应用。
背景技术
婴儿时期是发病率和死亡率都较高的特殊阶段,此时胃肠屏障功能不健全、机体免疫力差、抵抗力弱,极易发生感染性疾病,若使用或长期使用抗生素可导致肠道菌群失调,使病原菌乘虚而入,发生感染性疾病甚至暴发流行病。
鼠伤寒沙门氏菌是引起人和畜禽共患的常见致病菌,也是引起腹泻的重要病原菌之一,且居沙门氏菌属感染之首,以婴幼儿发病率高,不时构成较大流行,直接威胁着婴幼儿的健康。
双歧杆菌是最早定植于婴儿肠道的原生菌,占母乳喂养儿肠道总细菌数量的50%,有些甚至高达70%。越来越多的研究发现,婴儿肠道中的优势双歧杆菌主要是短双歧杆菌、长双歧杆菌、婴儿双歧杆菌等,其总数量与腹泻、过敏、特应性皮炎的发生率呈负相关。
鉴于此,筛选自健康婴儿、降低鼠伤寒沙门氏菌感染风险的益生菌及其相关产品的开发,将对保障婴幼儿健康、降低医疗支出具有重要意义。
发明内容
有鉴于此,本发明的目的在于提供一株短双歧杆菌YH68及其在降低鼠伤寒沙门氏菌感染风险产品中的应用,所述短双歧杆菌YH68可通过破坏鼠伤寒沙门氏菌的细胞膜完整性、下调鼠伤寒沙门氏菌主要毒力因子的mRNA转录水平等,从而抑制鼠伤寒沙门氏菌。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一株短双歧杆菌(Bifidobacterium breve)YH68,所述短双歧杆菌YH68保藏在中国微生物菌种保藏管理委员会普通微生物中心,生物保藏编号为CGMCCNO.14096。
本发明还提供了上述短双歧杆菌YH68在制备降低鼠伤寒沙门氏菌感染风险的食品、补充剂或药品中的应用。
优选的,所述预防和/或治疗表现为:下调所述鼠伤寒沙门氏菌致病相关毒力基因的表达和破坏所述鼠伤寒沙门氏菌的细胞膜的完整性。
优选的,所述鼠伤寒沙门氏菌致病相关毒力基因包括:编码侵袭蛋白的invF基因、编码入侵宿主细胞的转录调控基因hilA、编码I型菌毛的结构蛋白基因fimA、编码效应蛋白的基因sipA和sipB。
优选的,所述食品、补充剂或药品中,所述短双歧杆菌YH68以活细胞冻干粉、非活细胞形式、发酵上清液或发酵产物的形式存在。
优选的,所述短双歧杆菌YH68的活细胞冻干粉中,有效活菌数为3.0×1010~5.0×1010CFU/g。
优选的,所述食品、补充剂或药品中,所述短双歧杆菌YH68的有效活菌数为106~109CFU/mL或106~109CFU/g。
本发明提供了一株短双歧杆菌YH68,所述短双歧杆菌YH68不含获得性耐药,食用安全;具有较高的氨肽酶、β-半乳糖苷酶和葡萄糖苷酶活性;具有较强的耐酸性能和耐胆汁盐性能。在本发明实施例中,所述短双歧杆菌YH68可破坏鼠伤寒沙门氏菌细胞膜的完整性;同时还能使鼠伤寒沙门氏菌编码侵袭蛋白的invF基因表达量下调近8倍,编码入侵宿主细胞关键的转录调控基因hilA、编码I型菌毛的结构蛋白基因fimA、以及编码效应蛋白以导致感染的基因sipA和sipB表达量下降幅度均超过了10倍。本发明所述短双歧杆菌YH68可用于制备降低鼠伤寒沙门氏菌风险的食品、补充剂或药物。
保藏说明
短双歧杆菌(Bifidobacterium breve),菌株编号为YH68,所述短双歧杆菌YH68于2017年05月4日保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,生物保藏编号为CGMCC NO.14096。
具体实施方式
本发明提供了一株短双歧杆菌(Bifidobacterium breve)YH68,所述短双歧杆菌YH68保藏在中国微生物菌种保藏管理委员会普通微生物中心,生物保藏编号为CGMCCNO.14096。
本发明所述短双歧杆菌YH68,其形态为多形态杆状,革兰氏染色阳性,不形成芽孢,接触酶阴性,氧化酶阴性,专性厌氧;它能利用核糖、半乳糖、葡萄糖、果糖、甘露醇、山梨醇、α-甲基-D-葡萄糖甙、麦芽糖、乳糖、蜜二糖、蔗糖、海藻糖、棉子糖、淀粉、肝糖和D-松二糖进行代谢活动;不能利用赤藓醇、D-阿拉伯糖、L-阿拉伯糖、D-木糖、L-木糖、阿东醇、β-甲基-D-木糖甙、甘露糖、山梨糖、鼠李糖、卫矛醇、肌醇、α-甲基-D-甘露糖甙、N-乙酰-葡萄糖胺、苦杏仁甙、熊果甙、七叶灵、水杨甙、纤维二糖、菊糖、松叁糖、木糖醇、龙胆二糖、D-来苏糖、D-塔格糖、D-岩糖、L-岩糖、D-阿糖醇、L-阿糖醇、葡萄糖酸钠、2-酮基-葡萄糖酸钠和5-酮基-葡萄糖酸钠。
本发明还提供了上述短双歧杆菌YH68在制备降低鼠伤寒沙门氏菌感染风险的食品、补充剂或药品中的应用。
在本发明所述应用中,所述短双歧杆菌YH68优选以活细胞冻干粉、非活细胞形式、发酵上清液或发酵产物的形式存在。本发明所述短双歧杆菌YH68的活细胞冻干粉中,有效活菌数优选为3.0×1010~5.0×1010CFU/g。本发明对所述冻干粉、非活细胞形式及其发酵上清液或发酵产物的制备方法并没有特殊限定,所述发酵上清液或发酵产物优选在MRS液体培养基中37℃厌氧培养后获得。
本发明所述降低鼠伤寒沙门氏菌感染风险优选表现为:下调所述鼠伤寒沙门氏菌致病相关毒力基因的表达和破坏所述鼠伤寒沙门氏菌的细胞膜的完整性。在本发明中,所述鼠伤寒沙门氏菌致病相关毒力基因优选包括:编码侵袭蛋白的invF基因、编码入侵宿主细胞的转录调控基因hilA、编码I型菌毛的结构蛋白基因fimA、编码效应蛋白的基因sipA和sipB。
本发明对所述食品、补充剂或药品的剂型并没有特殊限定,优选的作为有效成分的短双歧杆菌YH68有效活菌数为106~109CFU/mL或106~109CFU/g。
下面结合实施例对本发明提供的短双歧杆菌YH68及其在制备降低鼠伤寒沙门氏菌感染风险产品中的应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
菌株分离鉴定
健康婴儿粪便样本采集与处理:经医院体格检查证实的11例健康初生婴儿,无腹泻及其他肠道疾病,母乳喂养。从出生后3h至194h婴儿刚换下的尿布上刮取新鲜粪便5~10g,装入无菌采集管内,采集管于厌氧盒保存。在采集管内加入50mL粪便稀释液,振荡制成匀浆,并依次10倍稀释至10-1至10-7。
双歧杆菌分离:采用双歧杆菌选择性培养基TPY,在GENbox厌氧培养系统(bioMerieux),培养36~48h,挑选不同形态特征的单菌落,转接至液体MRS培养基,37℃厌氧培养48h,连续转接3次可用于实验或冷冻保藏。
菌落形态观察和生理生化鉴定:通过菌落特征、菌体形态、革兰氏染色、糖发酵、过氧化氢酶等初步鉴定多个短双歧杆菌分离菌株。短双歧杆菌YH68菌落形态及生理生化试验结果如下:形态为多形态杆状,革兰氏染色阳性,不形成芽孢,接触酶阴性,氧化酶阴性,专性厌氧。其糖代谢谱结果见表1:
表1短双歧杆菌YH68糖代谢谱
注:“+”表示阳性,“-”表示阴性
由表1可知,所述短双歧杆菌YH68能利用核糖、半乳糖、葡萄糖、果糖、甘露醇、山梨醇、α-甲基-D-葡萄糖甙,麦芽糖、乳糖、蜜二糖、蔗糖、海藻糖、棉子糖、淀粉、肝糖和D-松二糖进行代谢活动;不能利用赤藓醇、D-阿拉伯糖、L-阿拉伯糖、D-木糖、L-木糖、阿东醇、β-甲基-D-木糖甙、甘露糖、山梨糖、鼠李糖、卫矛醇、肌醇、α-甲基-D-甘露糖甙、N-乙酰-葡萄糖胺、苦杏仁甙、熊果甙、七叶灵、水杨甙、纤维二糖、菊糖、松叁糖、木糖醇、龙胆二糖、D-来苏糖、D-塔格糖、D-岩糖、L-岩糖、D-阿糖醇、L-阿糖醇、葡萄糖酸钠、2-酮基-葡萄糖酸钠和5-酮基-葡萄糖酸钠。
实施例2
菌株筛选
1.耐药谱
基于欧盟对人和动物用的细菌最小抑菌浓度的限制(Guidance on theassessment of bacterial susceptibility to antimicrobials of human andveterinary importance,EFSA Journal,2012,10(6):2740),采用E-test药敏试剂盒(法国梅里埃)测定短双歧杆菌YH68对欧盟规定的9种抗生素的最小抑菌浓度(MIC,mg/L),结果如表2所示:
表2.短双歧杆菌YH68对欧盟规定的9种抗生素的最小抑菌浓度
由表2可知,所述短双歧杆菌YH68对9种抗生素的MIC与欧盟规定的一致,表明其除天生对卡那霉素有抗性外,不含获得性耐药,可安全食用。
2.有益酶活
采用API ZYM试剂盒(法国梅里埃公司)对所述短双歧杆菌YH68的酶活进行半定量分析。根据试剂盒操作说明,收集MRS厌氧培养液中生长的菌体细胞,用无菌水制备菌悬液用于下一步的酶活分析。加入菌体和试剂后,试剂条的色泽深度由浅到深分为0~5共6个等级,0相当于阴性反应,5为最强的反应。由色泽深度可知近似的菌体酶活,色泽1~5相当于释放的酶活浓度分别为5nmoles,10nmols,20nmols,30nmols和40nmols,结果如表3所示:
表3.API ZYM试剂盒酶活分析(用色泽0~5表示酶活)
酶 | 短双歧杆菌YH68 |
亮氨酸氨肽酶 | 5 |
β-半乳糖苷酶 | 5 |
α-葡萄糖苷酶 | 5 |
由表3可知,短双歧杆菌YH68具有极高的氨肽酶、葡萄糖苷酶和β-半乳糖苷酶活性。表明短双歧杆菌YH68能够分解蛋白质、乳糖、葡萄糖或葡萄糖类低聚糖等,有利于蛋白质和糖类的消化吸收。
3.耐酸性能
短双歧杆菌YH68于MRS液体培养基中37℃厌氧培养16小时后,于4℃、2500rpm下离心10min,收集菌体,磷酸盐缓冲液(PBS)洗涤后重悬于PBS中,取1.0mL细胞重悬液至pH为3.0的9.0mL无菌PBS中,37℃处理0、3h后进行活菌计数,计算存活率=3h的活菌数/0h的活菌数×100%。结果显示,短双歧杆菌YH68于pH3.0处理3h后的存活率仍有95%,表明YH68有较强的耐酸性能。
4.耐胆汁盐性能
短双歧杆菌YH68于MRS液体培养基中37℃厌氧培养16小时后,于4℃、2500rpm下离心10min,收集菌体,磷酸盐缓冲液(PBS)洗涤后重悬于PBS中,取1.0mL细胞重悬液至0.3%(w/v)胆盐(Bile salts,Merk)浓度的9.0mLPBS缓冲液中,于37℃处理3小时前后进行活菌计数分析,计算存活率=3h的活菌数/0h的活菌数×100%。结果显示,短双歧杆菌YH68于0.3%胆盐处理3h后的存活率仍有90%,表明YH68有较强的耐胆汁盐性能。
5.发酵性能
用接种环将短双歧杆菌YH68从斜面上挑取二环至装有50mL MRS液体培养基中,37℃厌氧培养24h,再以5%接种量接入50mL MRS液体培养基中,37℃厌氧培养72h,即得短双歧杆菌YH68发酵液,发酵液中活菌数大于3.0×108CFU/mL,表明其具有良好的发酵性能,可实现工业化生产。
6.菌粉制备
1)离心:将上述发酵液离心分离获得菌泥;
2)冷冻干燥:按水量为菌泥重量的6-15倍,将所述菌泥与水和冻干载体混合,冷冻干燥制成冻干活菌粉。冻干菌粉的活菌数为3.0×1010~5.0×1010CFU/g。
实施例3
采用琼脂斑点试验法,评估短双歧杆菌活菌对鼠伤寒沙门氏菌的抑制作用:
1)鼠伤寒沙门氏菌(S..typhimurium)ATCC 14028,购买自美国菌种保藏中心(ATCC),将ATCC 14028接种于新鲜的LB培养基中,生长至对数期时再次转接于新鲜培养基,传代3次后,将菌液与25%(v/v)的甘油以等体积混合后存放在-80℃冰箱中。活化时将冻存的菌液解冻后1%(v/v)接种于LB培养基中,在恒温摇床中约45°角倾斜放置,37℃、150r/min摇床震荡培养12h,备用。
2)将短双歧杆菌YH86用MRS液体培养基于37℃厌氧培养24h,发酵液中活菌数大于1.0×106CFU/毫升。
3)取上述2)培养好的活菌液0.1毫升点接于MRS固体平板上,37℃厌氧培养24h,每个平板平行三个点。
4)取200μLATCC 14028菌液与7mL软琼脂混匀,倒在上述3)的MRS平板上(注意软琼脂保持在45~50℃,不可过热或过冷),37℃好氧培养24h。
5)观察抑菌结果并用游标卡尺测量抑菌圈的直径,结果见显示短双歧杆菌YH68可以抑制鼠伤寒沙门氏菌活性,抑菌圈直径为21.0±0.45mm。
实施例4
用牛津杯试验法评估短双歧杆菌发酵产物对鼠伤寒沙门氏菌的抑制作用:
1)吸取0.3mL实施例3中培养好的ATCC 14028菌浓度约为1.0×106CFU/mL的指示菌悬液,无菌涂布棒涂布于LB固体培养基平板,涂布均匀,合上皿盖,在20℃培养箱中固定1h。
2)取出上述平板,用镊子将牛津杯小心地放置于平板上,取实施例3中短双歧杆菌YH68上清液200μL注入杯内,以MRS液体培养基(pH 3.6)作为阴性对照。
3)先在4℃冰箱扩散4h,然后将平板转移至37℃培养箱培养,24h后观察抑菌结果并用游标卡尺测量抑菌圈的直径,结果显示,所述短双歧杆菌YH68的发酵上清液可以抑制鼠伤寒沙门氏菌活性,抑菌圈直径为22.7±0.49mm。
实施例5
短双歧杆菌YH86通过破坏鼠伤寒沙门氏菌细胞膜的完整性抑制鼠伤寒沙门氏菌毒力
收集培养至对数生长期的鼠伤寒沙门氏菌细胞,用0.1mol/L pH 7.0的PBS缓冲液反复洗涤3次后,重悬于无菌水中,制备成菌悬液(106CFU/毫升),并向其中加入适量体积的YH68发酵上清液,以加入等体积无菌水的菌悬液作为空白对照,37℃恒温培养6h。分别在0、3h时间点取出上述2组菌悬液5mL,将样品进行离心(8000×g、10min、4℃)获得上清液,用电导率仪对上清液进行电导率的测定,结果如表4所示:
表4短双歧杆菌YH68处理3h对鼠伤寒沙门氏菌上清液电导率的影响
细胞膜是细菌的保护屏障,如果打破保护屏障,细胞内部电解质(如K+、Na+等)渗漏到细胞外的培养液中,使培养液的电导率上升。由表4可知,YH68处理后的电导率显著上升,表明其破坏了鼠伤寒沙门氏菌细胞膜的完整性,从而抑制其毒力。
实施例6
短双歧杆菌YH86显著下调鼠伤寒沙门氏菌主要毒力因子的mRNA转录水平,从而抑制鼠伤寒沙门氏菌毒力
用试剂盒提取实施例5中的2组鼠伤寒沙门氏菌总RNA,用DNase去除污染的基因组DNA。将总RNA逆转录合成cDNA,逆转录体系:总RNA1μg,Oligo dT 1μL,5×Buffer4μL,dNTPs(10mmol/L)2μL,RNase Inhibitor1μL,ReverTraAce 1μL,用ddH20补足至20μL。逆转录条件:65℃,5min;37℃,15min;98℃、5min。将RNA和逆转录的cDNA分别于-80℃和-20℃保存备用。
采用荧光定量PCR方法通过特异性引物来检测鼠伤寒沙门氏菌SPI-1相关基因的表达量,其中引物由Premier 5.0软件设计,由华大基因合成,引物序列如表5所示:
表5实时荧光定量RT-PCR引物
反应体系:cDNA 1μL,SYBR PremixExTaq(2×)7.5μL,ForwardPrimer0.3μL,ReversePrimer 0.3μL,ddH2O 5.9μL。以鼠伤寒沙门氏菌的16S rRNA作为内参,反应条件为:95℃,10min;95℃,30s、54℃,30s、72℃,30s循环40次。通过2-ΔΔCt方法对数据进行处理,将空白组相关基因的表达量标准化为1,确定YH68处理组基因的相对表达量。结果如表6所示:
表6主要毒力基因的相对表达量
由表6可知,YH68处理组与空白对照组相比,编码侵袭蛋白的invF基因表达量下调了近8倍,编码入侵宿主细胞关键的转录调控基因hilA、编码I型菌毛的结构蛋白基因fimA、以及编码效应蛋白以导致感染的基因sipA和sipB表达量下降幅度均超过了10倍。表明YH68可显著下调鼠伤寒沙门氏菌致病相关毒力基因的表达,从而抑制鼠伤寒沙门氏菌毒力。
本发明提供了一株短双歧杆菌YH68及其在降低鼠伤寒沙门氏菌感染风险中的应用,所述短双歧杆菌YH68可通过破坏鼠伤寒沙门氏菌细胞膜的完整性以及下调编码侵袭蛋白的invF基因表达量,下调编码入侵宿主细胞关键的转录调控基因hilA、编码I型菌毛的结构蛋白基因fimA、以及编码效应蛋白以导致感染的基因sipA和sipB的表达量,从而抑制鼠伤寒沙门氏菌活性,可用于制备预防和/或治疗鼠伤寒沙门氏菌引起疾病的食品、补充剂或药物。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.短双歧杆菌(Bifidobacterium breve)YH68在制备降低鼠伤寒沙门氏菌感染风险的药品中的应用,所述短双歧杆菌YH68保藏在中国微生物菌种保藏管理委员会普通微生物中心,生物保藏编号为CGMCC NO.14096。
2.根据权利要求1所述应用,其特征在于,所述降低鼠伤寒沙门氏菌感染风险表现为:下调所述鼠伤寒沙门氏菌致病相关毒力基因的表达和破坏所述鼠伤寒沙门氏菌的细胞膜的完整性。
3.根据权利要求2所述应用,其特征在于,所述鼠伤寒沙门氏菌致病相关毒力基因包括:编码侵袭蛋白的invF基因、编码入侵宿主细胞的转录调控基因hilA、编码I型菌毛的结构蛋白基因fimA、编码效应蛋白的基因sipA和sipB。
4.根据权利要求1所述应用,其特征在于,所述药品中,所述短双歧杆菌YH68以活细胞冻干粉或发酵产物的形式存在。
5.根据权利要求4所述应用,其特征在于,所述短双歧杆菌YH68的活细胞冻干粉中,有效活菌数为3.0×1010~5.0×1010CFU/g。
6.根据权利要求1所述应用,其特征在于,所述药品中,所述短双歧杆菌YH68的有效活菌数为106~109CFU/mL或106~109CFU/g。
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