CN116326684A - 一种寡糖组合物及其在调节动物肠道代谢物中的用途 - Google Patents
一种寡糖组合物及其在调节动物肠道代谢物中的用途 Download PDFInfo
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- CN116326684A CN116326684A CN202111597585.6A CN202111597585A CN116326684A CN 116326684 A CN116326684 A CN 116326684A CN 202111597585 A CN202111597585 A CN 202111597585A CN 116326684 A CN116326684 A CN 116326684A
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- oligosaccharide
- oligosaccharide composition
- intestinal
- algin
- animal
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Abstract
本发明涉及调节肠道代谢组合物,具体的说是一种寡糖组合物及其在调节动物肠道代谢物中的用途。寡糖组合物为按质量比为1:1的菊芋果寡糖和酶解获得褐藻胶寡糖。本发明的寡糖组合物可优化肠道菌群组成,调节所述动物肠道代谢物水平。
Description
技术领域
本发明涉及调节肠道代谢组合物,具体的说是一种寡糖组合物及其在调节动物肠道代谢物中的用途。
背景技术
肠道微生物负责将动物饮食中未经消化和未经吸收的组分转化为数千种生物活性代谢物。这些代谢物进而与宿主的局部和全身生理学相互作用。
在一些情况下,所产生的代谢物对宿主是有益的。例如,短链脂肪酸(SCFA)对消化道是有益的;其作为结肠细胞的能源物质可改善肠道功能并且保护肠道屏障。但由于其挥发性及特殊气味等原因,很难作为饲料添加剂配制成饲料。
益生元是不易消化的碳水化合物,它能够抵抗肠道酶的水解作用,但可由肠道有益微生物发酵,促进有益微生物生长,并促进短链脂肪酸(如乙酸,乳酸,丁酸)等代谢产物。
由于益生菌对底物偏好性差异,单一益生元往往只能选择性地促进某种或某几种益生菌,不利于肠道菌群微生物多样性。
菊芋是一种多年宿根性草本植物,其块茎中富含淀粉及菊糖等多糖。菊糖是以果糖为单元的聚合物,通过β(2–1)键连接,其中一个末端通过α(1–2)连接一个葡萄糖。
菊糖可在菊粉酶的作用下降解形成低聚果糖或果寡糖。果寡糖是益生菌优先利用碳源,它能促进有益菌的生长,抑制病原微生物。除了益生元的作用,经常和充足地摄入果寡糖,还可对预防胃肠道疾病、肥胖、腹泻等问题有益。
但单一的果寡糖作为益生元也存在很多局限性。例如,如文献(Pedersen etal.Gut hormone release and appetite regulation in healthy non-obeseparticipants following oligofructose intake.A dose-escalationstudy.Appetite.2013,44–53)所述,低聚果糖调节胃口和肠道荷尔蒙依赖于高剂量摄入。又如文献(Burokas et al.Targeting the Microbiota-Gut-Brain Axis:PrebioticsHave Anxiolytic and Antidepressant-like Effects and Reverse the Impact ofChronic Stress in Mice.Biol Psychiatry.2017 82(7):472-487.)所述,果寡糖主要是选择性地促进双歧杆菌的增殖,因此对肠道菌群和短链脂肪酸的调节具有局限性。
褐藻胶是海带的主要成分之一,是由α-L-甘露糖醛酸(M单元)和β-D-古罗糖醛酸(G单元)通过α-1,4糖苷键链接形成的一种高聚合度的线性多糖,它的提取物通常以纳盐形式存在,又称海藻酸钠。海藻酸钠作为增稠剂、稳定剂和乳化剂等广泛应用于食品和医药行业。同时,海藻酸钠还具有抗高血压、高血脂等生物活性。
由于褐藻胶黏度大、水溶性差、不易吸收,在医药领域的应用受到一定限制。
发明内容
本发明要解决的技术问题在于提供一种寡糖组合物及其在调节动物肠道代谢物中的用途。
为实现上述目的,本发明采用技术方案为:
一种寡糖组合物,寡糖组合物为按质量比为1:1的菊芋果寡糖和酶解获得褐藻胶寡糖。
所述菊芋寡糖为来源于菊芋在菊糖酶作用下的酶解产物;所述褐藻胶寡糖为来源于海带褐藻胶在褐藻胶裂解酶作用下的酶解产物。
所述菊芋寡糖聚合度为2-6;所述褐藻胶寡糖聚合度为2-10。
一种寡糖组合物的应用,所述寡糖组合物在调节动物肠道代谢物中的用途。
所述寡糖组合物在调节动物肠道代谢物短链脂肪酸中的用途。
所述的动物是小鼠、陪伴动物、家禽或家畜。
本发明的有益效果:
本发明寡糖组合物能够调节动物肠道促进短链脂肪酸(如乙酸,丙酸,丁酸)等代谢产物的产生,以及短链脂肪酸的丰度水平,同时具有改善肠道菌群结构,调节肠道微生态,促进机体健康的作用。
附图说明
图1为本发明实施例提供的实施例1鉴定酶法制备的菊芋果寡糖薄的聚合度的薄层层析色谱图。
图2为本发明实施例提供的实施例1鉴定酶法制备的褐藻胶果寡糖聚合度的薄层层析色谱图。
图3为本发明实施例提供的与葡萄糖相比,肠道拟杆菌利用寡糖时,发酵产物的变化。主要发酵产物从乳酸变成了乙酸。
图4为本发明实施例提供的寡糖代谢偶联的转录调控,解释了寡糖代谢促进短链脂肪酸产生的分子基础。
图5为本发明实施例提供的实施例2寡糖组合物对小鼠体重指标的影响,结果表明寡糖处理对小鼠生理状态有调控过程。
图6为本发明实施例提供的寡糖处理对小鼠的肠道菌群的调控。
图7展示了寡糖及寡糖组合物处理对小鼠肠道代谢物组的重塑。
图8为本发明实施例提供的不同处理组差异代谢物的代谢通路。
图9为本发明实施例提供的不同处理组短链脂肪酸的浓度。
具体实施例
下面结合具体实施例对上述实施步骤作出详细说明。
实施例1:菊芋果寡糖和褐藻胶寡糖的聚合度鉴定
1.菊芋果寡糖的聚合度鉴定
将酶法获得菊芋果寡糖(Wang Da et al.A one-step bioprocess forproduction of high-content fructo-oligosaccharides from inulin byyeast.Carbohydr Polym.2016,151:1220-1226)通过薄层层析法(TLC)进行分析以表征寡糖聚合度:将菊芋果寡糖制品配成0.2%的水溶液,取0.5-1μL样品点到F254 TLC板上,同时以纤维寡糖为标准品。在TLC展层液(正丁醇:甲酸:水(2:1:1v/v))中分离产物。层析板晾干后,喷洒苔黑酚显色液,放置于105℃下显色5min。鉴定结果如图1所示。
由图1结果表明:酶解法制备果寡糖的主要成分为二糖、三糖、四糖、五糖和六糖。
2.褐藻胶寡糖的聚合度鉴定
1)褐藻胶寡糖的获得:
优选的,所述褐藻胶寡糖可按如下步骤制备:
将海藻酸钠粉末溶解,配成0.5%的水溶液;
按照海藻酸钠粉末质量的1%加入内切型褐藻胶裂解酶(见Wang Bing etal.Substitution of one calcium-binding amino acid strengthens substratebinding in a thermophilic alginate lyase.FEBS Lett.2018 Feb;592(3):369-379.),在60℃度条件下,进行酶解;
在酶解液加入终浓度为70%乙醇,使酶解所得寡糖沉淀,离心收集沉淀,将所得沉淀冷冻干燥后即为褐藻胶寡糖粉末。
2)聚合度鉴定:
将褐藻胶寡糖制品配成0.2%的水溶液,取0.5-1μL样品点到F254 TLC板上,同时以聚合度2-6不饱和褐藻胶寡糖为标准品。在TLC展层液(正丁醇:甲酸:水(2:1:1v/v))中分离产物。层析板晾干后,喷洒苔黑酚显色液,放置于105℃下显色5min。鉴定结果如图2所示。
由图2结果表明:酶解法制备褐藻胶寡糖的主要成分为三糖至八糖,比较于化学酸解法,酶解法制备的褐藻胶寡糖在糖链的非还原端残基上形成一个不饱和双键,可促进特异性利用不饱和寡糖的益生菌增殖,生物活性更优。
将上述获得菊芋果寡糖与褐藻胶寡糖按照质量比为1:1。
实施例2:褐藻胶寡糖促进肠道益生菌产生短链脂肪酸
EGF培养基(pH 7.6,1L):2.4g Lab-Lemco powder,10g proteose peptone No.3,5g酵母提取物,4g Na2HPO4,5g葡萄糖,0.5g可溶性淀粉,0.5g半胱氨酸盐酸盐,1mg刃天青,10%马血。
S2无机盐培养基(pH 7.4-7.6,1L):50mM KH2PO4(pH 7.4-7.6),15mM NaCl,8.5mM(NH4)2SO4,6mM L-半胱氨酸,1.9μM羟高铁血红素,200μM L-组氨酸,1倍的维生素混合液和1倍微量无机盐混合液(母液配制如下文),使用前加入0.5%的葡萄糖或0.5%上述实施例制备获得褐藻胶寡糖作碳源。维生素混合液,羟高铁血红素和L-组氨酸分别配成100倍母液,过滤除菌后4℃保存,培养基使用前加入。
维生素混合液(100倍,mg L-1):维生素B12,0.5;维生素K3,100;生物素,0.2;叶酸,0.2;盐酸吡哆醇,10;盐酸硫胺素,5;核黄素,5;烟碱酸,5;泛酸钙,5;对氨基苯甲酸,5;硫辛酸,5。
微量无机盐组成(100倍母液,g L-1):EDTA,0.5;CaCl2,0.1;NaCl,1;MgSO4.7H2O,3;MnSO4.H2O,0.5;ZnSO4.7H2O,0.1;CuSO4.5H2O,0.01;H3BO3,0.01;Na2MoO4.2H2O,0.01;NiCl2.6H2O,0.02;CoCl2,0.1;FeSO4.7H2O,0.1。
发酵产物分析方法:高效液相色谱法(HPLC)。仪器:Agilent HPLC 1260(示差折光)。色谱条件:色谱柱:Aminex HPX-87H Column;流动相:5mM硫酸;流速:0.5mL/min;进样量:10μL;柱温:55℃;示差折光检测器。样品制备:发酵液4℃,12000rpm离心5min,取上清液适当稀释后,用0.22μm微孔滤膜过滤后进行HPLC分析。
肠道菌Bacteroides clarus JCM 10256购自日本菌种库JCM。菌种首先在EGF培养基中活化,37℃厌氧培养。
将复苏后的菌株接种到含有葡萄糖或褐藻胶寡糖的S2培养基中,待生长至指数中期后收集菌体,用于转录组测序。同时,培养基上清用于发酵产物分析。如图3所示,利用寡糖和葡萄糖时,B.clarus的主要发酵产物含乳酸,乙酸和琥珀酸。发酵葡萄糖时,产物主要是乳酸;发酵寡糖时,主要产物变为乙酸。
转录组测序分析发现,相比于以葡萄糖为碳源,利用寡糖时,共有589个基因显著变化。通过基因功能分析及代谢通路构建可知(图4),相比于葡萄糖,肠道菌对寡糖的发酵可同时调控碳代谢和氨基酸的代谢。碳代谢通路中,可上调产短链脂肪酸(乙酸和丁酸)相关通路。
实施例3:寡糖组合物对动物的施用
对小鼠接受寡糖组合物,以确定寡糖制剂对动物的生长性能的影响。
将上述将上述获得菊芋果寡糖与褐藻胶寡糖按照质量比为1:1混合作为寡糖组合物。
从济南鹏悦实验动物公司购得七周大的C57BL/6J雄性小鼠24只,将小鼠饲养在温度和湿度可控的房间中,进行12小时的明暗循环,并提供足够的可自由获取的食物和水。
实验开始前,小鼠在实验室条件下驯化7天后,随机分组,每组8只。对照组(NC)组给与常规食物和自来水,
褐藻胶寡糖组(AOS)给与常规食物以及含有0.1wt%褐藻胶寡糖的自来水,食物和水足量且可自由获取。
寡糖组合物组(FOS+AOS)给与常规食物以及含有0.1wt%寡糖组合物的自来水,食物和水足量且可自由获取。
实验开始当天记录小鼠的原始体重,并在实验开始后每两天记录一次体重,并观察小鼠的粪便,健康状况以及垫料是否结块等状况(参见图5)。
在实验第14天,记录当天体重后,通过CO2窒息法对小鼠实施安乐死。之后使用标准解剖方法提取大肠内容物,将内容物分装至两个1.5mlEP管中,并在液氮中速冻。
从小鼠体重结果看,对照组体重一直稳定上升,至实验结束时,体重增长率为14.4%;褐藻胶寡糖处理组在实验进行的前7天,体重稳定增长,且增长率略高于对照组;实验第8-12天,体重出现停滞;至第14天时,体重恢复增长,达到与NC组一致的增长率。寡糖组合物处理组在实验进行的第4天开始,体重急剧下降,至第8天,最大体重下降率达16%,推测此阶段为肠道菌群调整期;此后,小鼠体重极速恢复,至第14天时,体重增长达到14%,与NC组一致。
整个实验过程中,三组小鼠习性正常,饮水饮食均正常。
整个实验过程中,三组小鼠粪便正常,均未出现肉眼可见的腹泻。
实施例4:对小鼠肠道微生物菌群的16S rRNA基因全长测序
对从实施例3获取的三组24只小鼠的样品,进行微生物组16S rRNA基因全长测序。将肠道内容物解冻,并且使用标准方法提取DNA。用引物27F和1492R扩增16S rRNA基因全长。根据PacBio 2kb文库制备方法构建文库,并在PacBio sequel系统上测序。
27F:5’-AGAGTTTGATCMTGGCTCAG-3’
1492R:5’-GGYTACCTTGTTACGACTT-3’
删除含有不明确碱基或长度<1250bp的序列后,使用SILVA参考数据库(V132)进行序列比对;使用Mothur的“cluster”命令(版本1.39.5)以97%的相似性生成可操作分类单元(OTU)。只有保留读数≥2的OTU。OTU聚类基于Ribosomal Database Project数据库。以OTU的相对丰度评估肠道微生物群(参见图6)。
由图6可见寡糖组合物可促进有益菌的增殖,且效果优于单一寡糖。
实施例5:靶向代谢物组分析小鼠肠道代谢物组
对从实施例3获取的三组24只小鼠的样品,进行靶向代谢物组分析。将肠道内容物解冻后,称取5mg样品,加入25μL水,用氧化锆珠匀浆3分钟,并加入120μL含同位素内标物的甲醇以提取代谢物。将20μL上清液转移至96孔板中。向每个孔中添加20μL新制备的衍生试剂3-硝基苯肼。将平板密封,并在30℃下进行衍生化60分钟。衍生化后,添加330μL预冷的50%甲醇溶液以稀释样品。然后将该板在-20℃下储存20分钟,然后在4℃下进行4000g离心30分钟。将135μL上清液转移到新的96孔板中,每个孔中有10μL内标物。
使用超高效液相色谱-串联质谱(UPLC-MS/MS)系统对代谢物进行定量。以下简要介绍了优化的仪器设置。UPLC系统采用C18 1.7μM制备柱(2.1×5mm)和C18 1.7μM分析柱(2.1×100mm),柱温设置为40℃,流动相为A=0.1%甲酸水溶液;B=乙腈/IPA(70:30)。
靶向代谢组结果表明,寡糖处理对小鼠肠道的脂肪酸、氨基酸、碳水化合物和短链脂肪酸水平(图7、图8)均有明显调控。其中短链脂肪酸中,乙酸、丁酸、丙酸、戊酸和异戊酸丰度都有上升(图9)。
虽然本文已经显示和描述了本发明的优选实施方案,但对于本领域技术人员而言,此类实施方案仅通过举例的方式来提供。本发明的所有方面并不限于本文所阐述的条件和变量的具体描述、配置或相对比例。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 (6)
1.一种寡糖组合物,其特征在于:寡糖组合物为按质量比为1:1的菊芋果寡糖和酶解获得褐藻胶寡糖。
2.按权利要求1所述的寡糖组合物,其特征在于:所述菊芋寡糖为来源于菊芋在菊糖酶作用下的酶解产物;所述褐藻胶寡糖为来源于海带褐藻胶在褐藻胶裂解酶作用下的酶解产物。
3.按权利要求1或2所述的寡糖组合物,其特征在于:所述菊芋寡糖聚合度为2-6;所述褐藻胶寡糖聚合度为2-10。
4.一种按权利要求1所述的寡糖组合物的应用,其特征在于:所述寡糖组合物在调节动物肠道代谢物中的用途。
5.按权利要求4所述的寡糖组合物的应用,其特征在于:所述寡糖组合物在调节动物肠道代谢物短链脂肪酸中的用途。
6.按权利要求4所述的寡糖组合物的应用,其特征在于:所述的动物是小鼠、陪伴动物、家禽或家畜。
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