CN113647630B - 海参硫酸多糖及其弱酸降解产物促进乳杆菌增殖中的应用 - Google Patents
海参硫酸多糖及其弱酸降解产物促进乳杆菌增殖中的应用 Download PDFInfo
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
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Abstract
本发明提供海参硫酸多糖及其弱酸降解产物在促进乳杆菌增殖中的应用,用于促进格氏乳杆菌(Lactobacillusgasseri)、约氏乳杆菌(Lactobacillusjohnsonii)和罗伊氏乳杆菌(Lactobacillusreuteri)的增殖以及形成生物膜。
Description
技术领域
本发明涉及乳杆菌增殖领域,更具体地说,涉及海参硫酸多糖及其弱酸降解产物促进乳杆菌增殖中的应用。
背景技术
乳酸菌具有多种生物活性,包括改善胃肠道功能、维护肠道内微生态平衡、提高机体免疫力和降低血清胆固醇等。乳酸菌包括乳杆菌(Lactobacillus)、乳球菌(Lactococcus)和链球菌(Streptococcus)等,其中乳杆菌是乳酸菌中重要的一类。乳杆菌伴随人类的成长,从婴儿粪便中发现乳杆菌组成单一,主要是格氏乳酸杆菌(Lactobacillus gasseri)或唾液乳杆菌(Lactobacillus salivarius),而成人粪便中的乳杆菌菌株主要是鼠李糖乳杆菌(Lactobacillus rhamnosus)、干酪乳杆菌(Lactobacillus casei)或副干酪乳杆菌(Lactobacillus paracasei)。因此,乳杆菌也被认为是益生菌的一种。
艰难梭状芽胞杆菌感染是引起医院相关性胃肠道疾病的原因之一,艰难梭菌感染患者通常住院时间长,而且往往会造成医院感染的大爆发。对于在复发性艰难梭菌感染治疗的过程中,存在给患者服用抗生素(例如万古霉素、甲硝唑等)并且联合粪菌移植的治疗方案。对于该种治疗的术后恢复,目前尚无特定的有效方案。
发明内容
本发明提供了提供海参硫酸多糖促进乳杆菌增殖的新用途,以及海参硫酸多糖及其弱酸降解产物在促进乳杆菌增殖中的用途。
优选的,所述海参硫酸多糖、所述弱酸降解产物促进格氏乳杆菌(Lactobacillusgasseri)、罗伊氏乳杆菌(Lactobacillus reuteri)的增殖。
优选的,所述弱酸降解产物促进所述格氏乳杆菌(Lactobacillus gasseri)和约氏乳杆菌(Lactobacillus johnsonii)的增殖。
优选的,所述海参硫酸多糖在体外培养中,促进罗伊氏乳杆菌(Lactobacillusreuteri)乳杆菌的增殖。
优选的,所述海参硫酸多糖、所述弱酸降解产物促进格氏乳杆菌(Lactobacillusgasseri)、约氏乳杆菌(Lactobacillus johnsonii)和罗伊氏乳杆菌(Lactobacillusreuteri)形成生物膜。
优选的,所述弱酸降解产物在50~500μg/mL浓度时,促进罗伊氏乳杆菌(Lactobacillus reuteri)形成生物膜。
一种权利要求1所述海参硫酸多糖的制备方法,包括以下步骤:
S1、将海参在90~100℃水中煮至蛋白质变性,沥干、制成块,冻干再置于0~10℃丙酮中浸泡12~48h,室温下晾干,得到冻干样品;
S2、取所述冻干样品,加入0.1~0.3mol/L且pH 6.0的乙酸钠缓冲溶液、5~15%(w/w)木瓜蛋白酶、2~6%(w/w)乙二胺四乙酸和1~5%(w/w)半胱氨酸,混合后,于50~60℃水浴振荡酶解20~30h,在4000~8000g,5~20min,室温条件下离心取上清液;
S3、向所述上清液中加入5~15%(v/v)氯化十六烷基吡啶溶液,室温下放置12~48h后,在4000~8000g,5~20min,室温条件下离心取沉淀;将所述沉淀溶解于3mol/L的NaCl-乙醇溶液中,再加入80~95%乙醇溶液,0~10℃放置24h,在4000~8000g,5~20min,室温条件下,离心取沉淀;
S4、用80%和95%乙醇分别洗2~3次,室温晾干,用蒸馏水溶解,用1000~4000Da的透析袋除盐,冻干后得海参硫酸多糖。
优选的,步骤S3中所述NaCl-乙醇溶液中NaCl与乙醇的比例为100:15v/v。
一种权利要求1所述弱酸降解产物的制备方法,包括以下步骤:
S1、取海参硫酸多糖,溶于的1.0~1.5mol/L三氟乙酸中,在100℃水解4小时,使海参多糖降解为单糖和寡糖;
S2、浓缩干燥后,按海参硫酸多糖与甲醇1:1.0~2.5m/v的比例添加甲醇,再次浓缩干燥至无明显液体,重复操作2~3次,以充分除去三氟乙酸;
S3、以4000~8000g离心取上清液,将沉淀继续洗涤离心1~2次后,再收集所有上清液,冻干后,得海参硫酸多糖弱酸降解产物。
本发明的有益效果:
海参硫酸多糖是海参中主要的活性成分之一。本专利研究发现海参硫酸多糖能够有效促进抗生素处理联合粪菌移植后肠道内乳杆菌增长,有效促进乳杆菌生物膜形成,有利于肠道菌群的恢复。海参多糖弱酸降解产物能够有效促进乳杆菌增殖,具有益生元作用。因此,海参硫酸多糖及其弱酸降解产物具有成为功能食品的应用潜力。
附图说明
图1是实施例2中海参硫酸多糖的弱酸降解产物中的单糖组成的选择离子色谱图;
图2是实施例2中海参硫酸多糖的弱酸降解产物中的寡糖的选择离子色谱图;
图3是实施例2中海参硫酸多糖的弱酸降解产物中的岩藻糖的一级质谱图;
图4是实施例2中海参硫酸多糖的弱酸降解产物中的氨基葡萄糖的一级质谱图;
图5是实施例2中海参硫酸多糖的弱酸降解产物中的葡萄糖醛酸的一级质谱图;
图6是实施例2中海参硫酸多糖的弱酸降解产物中的氨基半乳糖的一级质谱图;
图7是实施例2中海参硫酸多糖的弱酸降解产物中的岩藻糖二糖的二级质谱图;
图8是实施例2中海参硫酸多糖的弱酸降解产物中的葡萄糖醛酸+氨基半乳糖二糖的二级质谱图;
图9是实施例2中海参硫酸多糖的弱酸降解产物中的岩藻糖+葡萄糖醛酸+氨基半乳糖三糖的二级质谱图;
图10是实施例3中抗生素处理小鼠后粪便中肠道菌群菌落数;
图11是实施例3中抗生素处理小鼠后,粪菌移植10天后小鼠粪便中肠道菌群菌落数;
图12是实施例3中海参硫酸多糖及其弱酸降解产物对鼠肝脏指数的影响;
图13是实施例3中海参硫酸多糖及其弱酸降解产物对鼠肾脏指数的影响;
图14是实施例3中海参硫酸多糖及其弱酸降解产物对鼠结直肠指数的影响;
图15是实施例3中海参硫酸多糖及其弱酸降解产物对小鼠粪便中乳杆菌属的相对含量的影响;
图16是实施例4中海参硫酸多糖及其弱酸降解产物和组分单糖对格氏乳杆菌菌株的生长影响;
图17是实施例4中海参硫酸多糖及其弱酸降解产物和组分单糖对罗伊氏乳杆菌菌株的生长影响;
图18是实施例4中海参硫酸多糖及其弱酸降解产物和组分单糖对约氏乳杆菌菌株的生长影响;
图19是实施例5中海参硫酸多糖及其弱酸降解产物对格氏乳杆菌菌株的生物膜形成的影响;
图20是实施例5中海参硫酸多糖及其弱酸降解产物对罗伊氏乳杆菌菌株的生物膜形成的影响;
图21是实施例5中海参硫酸多糖及其弱酸降解产物对约氏乳杆菌菌株的生物膜形成的影响。
具体实施方式
下面结合具体实施例对本发明做进一步说明。下述实施例所用原料及试剂均可由市场购得。
一种海参硫酸多糖的制备方法,包括以下步骤:
S1、将海参在90~100℃水中煮至蛋白质变性,沥干、制成块,冻干再置于0~10℃丙酮中浸泡12~48h,室温下晾干,得到冻干样品;
S2、取所述冻干样品,加入0.1~0.3mol/L且pH 6.0的乙酸钠缓冲溶液、5~15%(w/w)木瓜蛋白酶、2~6%(w/w)乙二胺四乙酸和1~5%(w/w)半胱氨酸,混合后,于50~60℃水浴振荡酶解20~30h,在4000~8000g,5~20min,室温条件下离心取上清液;
S3、向所述上清液中加入5~15%(v/v)氯化十六烷基吡啶溶液,室温下放置12~48h后,在4000~8000g,5~20min,室温条件下离心取沉淀;将所述沉淀溶解于3mol/L的NaCl-乙醇溶液中,再加入80~95%乙醇溶液,0~10℃放置24h,在4000~8000g,5~20min,室温条件下,离心取沉淀;所述NaCl-乙醇溶液中NaCl与乙醇的比例为100:15v/v;
S4、用80%和95%乙醇分别洗2~3次,室温晾干,用蒸馏水溶解,用1000~4000Da的透析袋除盐,冻干后得海参硫酸多糖。
一种弱酸降解产物的制备方法,包括以下步骤:
S1、取海参硫酸多糖,溶于的1.0~1.5mol/L三氟乙酸中,在100℃水解4小时,使海参多糖降解为单糖和寡糖;
S2、浓缩干燥后,按海参硫酸多糖与甲醇1:1.0~2.5m/v的比例添加甲醇,再次浓缩干燥至无明显液体,重复操作2~3次,以充分除去三氟乙酸;
S3、以4000~8000g离心10~30min,取上清液,将沉淀继续洗涤离心1~2次后,再收集所有上清液,冻干后,得海参硫酸多糖弱酸降解产物。
实施例1
本实施例用以制备海参硫酸多糖及其弱酸降解产物。
具体方法如下:
将海参洗净、水煮、沥干、剪成小块,然后冻干得到样品。将冻干样品置于4℃丙酮中浸泡24h,室温下晾干。以1.0g冻干样品为例,加入30mL的0.1mol/L乙酸钠缓冲溶液(pH6.0)、100mg木瓜蛋白酶、48mg乙二胺四乙酸和18mg半胱氨酸,涡旋混合,于60℃水浴振荡酶解24h,反应混合物离心(6000g,15min,室温)取上清液。向上清液加入1.6mL的10%氯化十六烷基吡啶溶液,室温下放置24h后,离心(8000g,15min,室温)取沉淀。沉淀溶解于15mL的3mol/L的NaCl-乙醇(100:15v/v)溶液中,再加入30mL的95%乙醇溶液,4℃放置24h,离心(8000g,15min,室温)取沉淀。并用30mL的80%和95%乙醇分别洗2至3次,室温晾干,蒸馏水溶解,用透析袋(3500Da)进行除盐,冻干,得海参硫酸多糖。
称取1.0g海参硫酸多糖,溶于50mL的1.3mol/L三氟乙酸中,在100℃水解4小时。浓缩干燥后,添加2.0mL甲醇,再离心浓缩至干燥,重复操作两次。反应结束,以8000g离心20分钟,取上清液,沉淀继续洗涤离心1-2次后,收集所有上清液。冻干后,得海参硫酸多糖的弱酸降解产物。
实施例2
本实施例用以确定实施例1所制备的海参硫酸多糖的弱酸降解产物的组成。
具体方法如下:
通过PMP衍生化法和高效液相色谱质谱联用检测海参多糖的弱酸降解产物的寡糖和单糖组成。取样品5.0mg,加入400μL氨水,400μL的PMP在70℃水浴锅中加热搅拌30分钟。离心浓缩至干燥后,加入500μL甲醇,再离心浓缩至干燥,重复操作两次。加入1%乙酸1.0mL和氯仿1.0mL,振荡5分钟后除去氯仿,重复萃取三次,水层作为供试液。将供试液稀释10倍后,过0.22μm的微孔滤膜。将准备好的样品进高效液相色谱质谱联用上机检测。使用C18色谱柱;流速为0.2mL/min;流动相A为20mM乙酸铵水溶液,流动相B为乙腈,比例为83:17。
如图1所示,岩藻糖(Fuc)、氨基葡萄糖(GlcN)、葡萄糖醛酸(GlcA)和氨基半乳糖(GalN)的摩尔比为12.3:0.4:0.5:1.0。如图2所示,寡糖组成分别有GlcA+GalN,Fuc+GlcA+GalN,Fuc+Fuc和Fuc+GalN,其摩尔比为8.5:2.7:3.6:1.0。通过图3-9,验证上述单糖、寡糖的一级和二级质谱图,确认结构正确。
实施例3
本实施例将上述实施例1所制得海参硫酸多糖及其弱酸降解产物进行饮食干预实验。
1.人肠道菌群接种液制备
收集志愿者粪便,要求为3个月内未服用过抗生素,饮食正常并且身体健康。取志愿者的粪便1.0g,溶解于10mL灭菌PBS溶液中(pH=7.2)。振荡混匀后,600×g,4℃离心5分钟,取上清液于新的灭菌离心管中。加入一定量的40%灭菌甘油,至溶液中甘油终浓度为20%。将溶液按1.0mL/管分装于灭菌的2.0mL离心管中,冷冻于-80℃冰箱中。
2.小鼠模型的构建
C57BL/6小鼠12只,SPF级,4周龄,雄性,购自辽宁长生生物技术股份有限公司。受试动物进入大连工业大学国家海洋食品工程技术研究中心动物实验室,6只/笼,20-25℃,RH 40~60%,每12小时切换照明,即每12h明暗交替照明;自由进食饮水,饮用水为中心制备的蒸馏水。待小鼠适应7天后,在饮水中加入万古霉素(0.045mg/mL),卡那霉素(0.4mg/mL),甲硝唑(0.215mg/mL),庆大霉素(0.035mg/mL)和粘菌素(850U/mL)连续服用5天后。随机收集小鼠粪便,装入灭菌的离心管中,加入1.0mL灭菌的PBS(pH=7.2),振荡混匀后,600×g,4℃离心5分钟,取上清液于新的灭菌离心管中。按照无菌检查法,接种于硫乙醇培养基(FT)和脑心浸液肉汤培养基(BHI)固体平板上,置于3种不同条件培养,分别是需氧,37℃;需氧,25℃;厌氧,37℃。如图10所示,当小鼠粪便细菌数量下降3个数量级,确定为抗生素处理成功。随后每天灌胃实验1中制备的人肠道菌群接种液0.2mL/只小鼠,连续灌胃14天后,按照上述无菌检查法,检查小鼠粪便的细菌数量。如图11所示,小鼠粪便细菌数量上升至和未处理组小鼠相同,判定为小鼠模型构建成功。
3.实验方法
3.1动物实验
将造模成功的12只小鼠随机分为两组,6只为正常组给予维持饲料,6只为实验组给予维持饲料并且每天灌胃300mg/kg小鼠体重的海参硫酸多糖。
每日观察小鼠体重和精神状态。灌胃10天后,采集各组小鼠新鲜粪便于无菌试管中,立即储存于-80℃冰箱,待后续菌群分析。试验周期结束,过夜禁食,眼底动脉丛取血后处死。精确分离肾脏、肝脏、盲肠和结直肠,洗净,吸干,称重。同时,取盲肠内容物,储存于-80℃冰箱。
3.2肠道菌群检测
收集来自小鼠的粪便样品,并通过PowerFecalTMDNA分离试剂盒(MO BIO)提取基因组DNA。通过1%琼脂糖凝胶电泳评估DNA的质量,然后纯化后用NanoDrop分光光度计测定其浓度。用特异于16S rRNA V3-V4区域的通用引物515F/806R扩增PCR产物,并使用QIAquick凝胶提取试剂盒进行纯化。使用Ion Plus Fragment Library Kit 48rxns生成测序文库,并在Qubit@2.0荧光计(Thermo Scientific)上进行评估。测序在诺和基因生物信息技术有限公司(中国北京)的Ion S5TMXL平台上进行。原始读数经过过滤,并根据条形码进行分离。在去除了嵌合序列后,将条形码和引物截短后,可获得纯净的读数。所有样品的有效标签均以97%的同一性归类为可操作的生物分类单位(OTU)。注释OTU的代表性序列以获得分类学信息,并使用直方图显示分类级别的每个样本的群落组成。使用QIIME软件(v1.9.1)和R软件(v2.15.3)来计算和显示OTU的丰度。
3.3数据处理
采用SPSS22.0统计学软件进行数据统计,数据结果以均值±标准偏差表示,组间数据显著性差异采用方差分析进行比较,显著性水平设为p<0.05。
4.实验结果
4.1各受试物对小鼠肾脏、肝脏和结直肠指数的影响
由图12-14可知,正常组和实验组小鼠的肾脏、肝脏和结直肠指数没有显著性差异。肾脏、肝脏和结直肠指数是由脏器重量或者肠道长度除以小鼠体重计算。
4.2海参硫酸多糖促进肠道菌群中乳杆菌属的增殖
如图15可知,通过16S rRNA测序结果发现,海参硫酸多糖显著促进乳杆菌的增殖,主要是格氏乳杆菌(Lactobacillus gasseri)和罗伊氏乳杆菌(Lactobacillus reuteri)。
从以上实验结果可以看出,本发明所述海参硫酸多糖对小鼠没有副作用,并且可以有效增加小鼠肠道中的乳杆菌相对含量,尤其是格氏乳杆菌(Lactobacillus gasseri)和罗伊氏乳杆菌(Lactobacillus reuteri)增殖最为显著。
实施例4
利用实施例1中的提取获得的海参硫酸多糖及其弱酸降解产物体外培养乳杆菌。
乳杆菌菌株(Lactobacillus gasseri,Lactobacillus reuteri和Lactobacillusjohnsonii)在MRS培养基上于37℃厌氧环境中生长,培养12小时。使用配备有BioLink软件包的自动生长曲线分析仪(Bioscreen C),新鲜培养的菌液按1%接种量,接种于含有10mg/mL岩藻糖(Fuc),氨基葡萄糖(GlcN),葡萄糖醛酸(GlcA),氨基半乳糖(GalN),N-乙酰氨基半乳糖(GalNAc),海参硫酸多糖(SCSP)或海参硫酸多糖的弱酸降解产物(o-SCSP)的200μL无糖MRS培养基中。将无糖MRS培养基设置为空白对照(Blank)。通过OD600nm监测菌株生长情况。
结果如图16-18所示,当o-SCSP成为培养基中唯一的碳源时,L.reuteri乳杆菌显著增殖,而对L.gasseri乳杆菌或L.johnsonii乳杆菌几乎没有效果。此时,由于组成海参硫酸多糖的单糖分别有岩藻糖(Fuc),氨基葡萄糖(GlcN),葡萄糖醛酸(GlcA),氨基半乳糖(GalN),N-乙酰氨基半乳糖(GalNAc)。结果发现,GlcN能够显著促进了除L.johnsonii乳杆菌之外的另外三株乳杆菌显著增殖。并且,GalNAc在促进乳杆菌增殖方面比GalN具有更强的效果。
综上所述,海参硫酸多糖在体外培养中,只能促进罗伊氏乳杆菌(L.reuteri)乳杆菌的增殖。海参多糖的弱酸降解产物可以显著促进格氏乳杆菌(L.gasseri)和约氏乳杆菌(L.johnsonii)的增殖。
实施例5
利用实施例1中的提取获得的海参硫酸多糖及其弱酸降解产物在体外促进乳杆菌生物膜的形成。
乳杆菌菌株(Lactobacillus gasseri,Lactobacillus reuteri和Lactobacillusjohnsonii)在MRS培养基上于37℃厌氧环境中生长,培养12小时,使用PBS(pH=7.2)调整OD600至10的7次方/mL菌液浓度。向灭菌的96孔板中添加上述菌液200mL/孔,培养基使用AOAC培养基(含SCSP浓度为0、12.5、50和100μg/mL)。96孔板于37℃培养24小时。丢弃培养基,用PBS清洗2次。加入200μL的0.1%结晶紫(溶解于异丙醇:甲醇:PBS=1:1:18)染色30分钟。丢弃溶液,用蒸馏水清洗3次后,于空气中晾干30分钟。每孔中添加200μL的30%冰醋酸溶解菌膜,测定OD570nm。
结果如图19-21所示,海参硫酸多糖(SCSP)在200μg/mL时,能够显著促进L.gasseri、L.johnsonii和L.reuteri乳杆菌形成生物膜。海参多糖的弱酸降解产物(o-SCSP)在200μg/mL时,能够促进L.reuteri乳杆菌形成生物膜。结果说明,o-SCSP促进乳杆菌形成生物膜的能力比SCSP降低24~40%。并且,SCSP促进乳杆菌在体外形成生物膜的能力是随浓度增长而增强的。
综上,海参硫酸多糖及其弱酸降解产物能够在体外促进乳杆菌形成生物膜。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (3)
1.一种海参硫酸多糖在体外促进格氏乳杆菌(Lactobacillus gasseri)、约氏乳杆菌(Lactobacillus johnsonii)和罗伊氏乳杆菌(Lactobacillus reuteri)形成生物膜的用途。
2.一种海参硫酸多糖弱酸降解产物在体外促进罗伊氏乳杆菌(Lactobacillus reuteri)形成生物膜的用途,其特征在于,所述弱酸降解产物在50~500μg/mL浓度时,促进罗伊氏乳杆菌(Lactobacillus reuteri)形成生物膜。
3.一种海参硫酸多糖弱酸降解产物在体外培养中促进格氏乳杆菌(Lactobacillus gasseri)、罗伊氏乳杆菌(Lactobacillus reuteri)增殖的用途。
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