CN114027510A - 一种蛋白核小球藻多糖混合物及其制备方法和作为新型益生元的应用 - Google Patents
一种蛋白核小球藻多糖混合物及其制备方法和作为新型益生元的应用 Download PDFInfo
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- CN114027510A CN114027510A CN202111390253.0A CN202111390253A CN114027510A CN 114027510 A CN114027510 A CN 114027510A CN 202111390253 A CN202111390253 A CN 202111390253A CN 114027510 A CN114027510 A CN 114027510A
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- chlorella pyrenoidosa
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Abstract
本发明公开了一种蛋白核小球藻多糖混合物及其制备方法和作为新型益生元的应用,本发明制备的CPP分子量(Mw)范围为10,000~600,000Da,总糖含量为45%~75%,葡萄糖醛酸(GlcA)含量为10%~30%,单糖组成为甘露糖(Man)、核糖(Rib)、鼠李糖(Rha)、GlcA、Glc、半乳糖(Gal)、木糖(Xyl)和阿拉伯糖(Ara)。制备方法为:水提醇沉,淀粉酶水解,除蛋白,透析。所得CPP水溶性好,无毒副作用,具有抵抗唾液、胃肠消化液的消化特性,能被粪便肠道微生物利用,促进有益菌群的增殖,抑制有害菌群的生长,促进肠道菌群产生短链脂肪酸,尤其能够促进产生大量的狄氏副拟杆菌(Parabacteroides distasonis),其发酵特征异于传统益生元,可作为益生元相关药物和食品添加剂改善或治疗肠道菌群紊乱相关疾病,应用于医药、食品等领域。
Description
技术领域
本发明属于医药生物技术领域,具体涉及一种蛋白核小球藻多糖混合物及其制备方法和作为新型益生元的应用。
背景技术
肠道菌群作为最重要和最复杂的人类微生态系统之一,具有重要的生理功能。肠道菌群紊乱与许多疾病的发生、发展密切相关,如肠道菌群丰度降低能够导致肥胖症、炎症、糖尿病、心血管疾病等的发生及恶化。合理膳食包括摄取更多“益生元”(Prebiotics)能够调节肠道微生物,从而治疗各种疾病、改善人体健康。传统的益生元如低聚果糖(FOS)、半乳聚糖(GOS)、低聚已麦芽糖(IMO)等能够促进乳酸菌(Lactobacillus)和/或双歧杆菌(Bifidobacterium spp.)的增殖,从而改善人体健康。但这些传统的益生元具有发酵特征方面的缺陷如发酵速率太快、调节的肠道微生物种类有限、产生有害代谢产物等(Yu etal.Carbohydr.Polym.2021,270:118377.)。随着人类健康需求的增加,新型益生元的市场需求也在日益增长。
小球藻具有许多优良特性,如易培养、生长速度快、耐不利生长条件、营养丰富、各种生物活性物质含量高等。大量研究表明,小球藻食用后表现出各种生物活性,如抗肿瘤、抗氧化、免疫调节、抗糖尿病等(Yuan et al.Int.J.Biol.Macromol.2020,163:2199–2209.)。因此,小球藻被联合国粮食及农业组织(FAO)列为“绿色健康食品”,有成为一种解决21世纪世界粮食问题的新型作物的巨大潜力(Torres-Tiji etal.Biotechnol.Adv.2020,41:107536.)。而多糖是小球藻最为丰富的活性物质之一。研究表明,小球藻多糖经口服后具有广泛的生物活性,如免疫调节和降血脂等。普遍认为多糖难以通过血脑屏障,小球藻多糖在口服后如何变化及发挥其活性鲜见研究报道。目前,蛋白核小球藻是小球藻中最为常见的物种之一,其作为功能性食品被广泛研究及利用,但鲜见小球藻多糖作为益生元改善肠道菌群,尤其促进狄氏副拟杆菌(Parabacteroidesdistasonis)生长的研究报道。
发明内容
鉴于上述内容,本发明从蛋白核小球藻中提取总多糖CPP,采用淀粉酶除去淀粉类多糖,得到本发明一种蛋白核小球藻多糖混合物,进一步研究发现该混合物能够明显增加肠道微生物的丰度,增加有益菌含量,尤其显著促进狄氏副拟杆菌(Parabacteroidesdistasonis)的增殖;并且能够抑制有害菌的生长,提高不饱和脂肪酸的浓度,具有独特的发酵特征。因此,本发明一种蛋白核小球藻多糖混合物作为新型益生元在调节肠道微生物、制备改善或治疗肠道菌群紊乱相关制剂中具有重要的应用价值。
本发明是通过下述技术方案实现的:
一种蛋白核小球藻多糖混合物,按照下述步骤制备获得:
步骤一、取蛋白核小球藻干粉,热水浸提或加木瓜蛋白酶酶解处理获得提取液;
步骤二、加入淀粉酶水解淀粉类多糖,乙醇和/或丙酮沉淀得到多糖提取物;
步骤三、透析、超滤或凝胶过滤除小分子杂质;
步骤四、最后减压冷冻干燥获得目标产物。
在上述技术方案中,在步骤一中,取蛋白核小球藻干粉置于反应釜中加水提取,料液比为1:10~1:30,85~95℃提取1~3次,每次1~4h,离心并合并所得提取液。
在上述技术方案中,在步骤二中,所述淀粉酶终浓度为0.01%~0.5%,溶液pH为5.5~7.5,酶解温度为40~70℃,酶解时间为1~4h,淀粉KI试纸监测直至水解液不变蓝为止。
在上述技术方案中,所述的一种蛋白核小球藻多糖混合物由5种不同分子量的多糖构成,分子量范围为10,000~650,000Da。
在上述技术方案中,所述的一种蛋白核小球藻多糖混合物的分子量范围优选为15,000~600,000Da。
在上述技术方案中,所述的一种蛋白核小球藻多糖混合物的总糖含量以苯酚硫酸法(葡萄糖(Glc)为标准品)检测为45%~75%,葡萄糖醛酸(GlcA)含量为10%~30%,不含蛋白质,易溶于水。
在上述技术方案中,所述的一种蛋白核小球藻多糖混合物包含的单糖组成为甘露糖(Man)、核糖(Rib)、鼠李糖(Rha)、GlcA、Glc、半乳糖(Gal)、木糖(Xyl)和阿拉伯糖(Ara),其摩尔比为(1.2±0.5):(0.6±0.3):(1.6±0.5):(1.8±0.5):(1.1±0.5):(3.0±0.5):(0.4±0.3):(2.3±0.5)。
一种蛋白核小球藻多糖混合物作为新型益生元在调节肠道微生物中的应用。
一种蛋白核小球藻多糖混合物作为新型益生元在口服制剂或食品添加剂或口服药物中的应用。
进一步地,所述一种蛋白核小球藻多糖混合物具有抵抗唾液、胃肠液的消化,能够被人肠道微生物酵解利用,提高人肠道微生物物种的丰度和多样性。明显增加有益菌如副杆菌属(Parabacteroides)、考拉杆菌属(Phascolarctobacterium)和拟杆菌属(Bacteroides)的水平,显著抑制大肠埃希菌-志贺菌属(Escherichia-Shigella)、梭菌属(Fusobacterium)和克雷白氏杆菌属(Klebsiella)等有害菌增殖。
进一步地,所述一种蛋白核小球藻多糖混合物能够促进短链脂肪酸(SCFAs)如乙酸、丙酸、丁酸、戊酸等的产生。
进一步地,所述一种蛋白核小球藻多糖混合物发酵特征异于传统益生元为CPP发酵速率,SCFAs水平变化趋势明显异于传统益生元如低聚果糖(FOS)、低聚已麦芽糖(IMO)等。
本发明具有如下有益效果:
1、本发明一种蛋白核小球藻多糖混合物提取时采用淀粉酶去除淀粉类多糖的化合物,避免了淀粉类物质对其益生元功效的不良影响。
2、本发明一种蛋白核小球藻多糖混合物具有新型益生元的发酵特征,能够促进有益菌的产生,尤其能够促进产生大量的具有治疗多发性硬化症及肥胖症等的狄氏副拟杆菌(Parabacteroides distasonis),该特征异于传统益生元。由于寻找新型益生元已成为通过调节肠道微生物治疗疾病及提高人体健康研究的重点方向,因此,本发明一种蛋白核小球藻多糖混合物对于研制新型益生元及食品添加剂具有重要的应用价值。
3、大多数传统益生元发酵速率太快,很容易被肠道微生物利用,产生大量的酸性代谢物,显著降低肠道环境的pH值,具有不良效果。本发明一种蛋白核小球藻多糖混合物不被人体胃肠液的消化酶消化,发酵速率缓慢增加,不饱和脂肪酸等代谢产物浓度也逐渐增大,其发酵特征异于传统益生元,有望成为新型益生元用于食品及药品。
附图说明
图1为蛋白核小球藻多糖混合物CPP的HPGPC检测谱图;
图2为CPP的1HNMR检测图谱;
图3为CPP体外模拟肠道菌群发酵的色谱图;
图4为CPP发酵时发酵液pH变化;
图5为CPP发酵后微生物组成的OUT水平分析结果;
图6为CPP发酵后微生物在门水平和属水平的相对丰度;
图7为CPP发酵后微生物在操作分类单元(OUT)水平的LEfSe分析。
对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,可以根据以上附图获得其他的相关附图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似改进,因此本发明不受下面公开的具体实施的限制。
实施例1:
蛋白核小球藻多糖混合物(CPP)的制备及理化性质
1.材料和方法
(1)材料
蛋白核小球藻(Chlorellapyrenoidosa),市售品;
单糖标准品Man、Ara、Gal、GalA、Glc、GlcA、Rib、Xyl、Rha、Fuc、木瓜蛋白酶、淀粉酶(50U/mg)、苯酚、硫酸、NaCl、NaOH、乙醇等所用试剂均为市售色谱纯或分析纯试剂。
(2)方法
蛋白核小球藻多糖混合物(CPP)制备:取蛋白核小球藻干粉30g,与蒸馏水按1:20(w/v)的比例混合,在90℃下提取3h。4500rpm离心后,在相同条件下再提取沉淀2次。合并上清液,加入淀粉酶比例为0.1%,pH 6.5,温度50℃,酶解时间2h,淀粉碘化钾试纸监测,直到淀粉碘化钾试纸不变蓝,沸水浴10min灭酶活,离心后的上清液加入95%食用酒精至终浓度为70%,4℃静置4h。离心得沉淀,复溶后用Sevag法除蛋白质。除蛋白的水溶液用透析袋(截留分子量为3.5kDa)于蒸馏水中透析3天。透析液浓缩、冻干得到CPP。
CPP理化性质分析:总糖含量采用苯酚硫酸法,D-Glc为标准品;糖醛酸含量测定采用羟基联苯法,D-GlcA为标准品;蛋白质含量测定采用考马斯亮蓝法(张惟杰,糖复合物生化研究技术(第二版),浙江:浙江大学出版社,1999,11-21)。分子量及分布采用高效凝胶色谱法(HPGPC)检测,色谱条件:岛津LC-2030C 3D Plus高效液相色谱仪,Shodex SB-804HQ(8.0mm ID×300mm)凝胶柱,示差折光检测器(RID),流动相为0.1M NaCl溶液,流速为0.5mL/min。单糖组成采用柱前衍生化HPLC检测,色谱柱为Eclipse Plus C18(4.6×250mm,5μm),流动相为0.1M磷酸盐缓冲液(pH 6.7)与乙腈的混合液(体积比为83:17),流速为1.0mL/min,柱温30℃,检测器为DAD,检测波长245nm,进样量20μL。NMR谱图采用AVANCEAV600核磁共振谱仪(600MHz)检测(瑞士Bruker公司,溶剂为D2O,温度25℃)。
2.结果:
CPP的HPGPC检测谱图见附图1,理化参数测定结果见表1;1HNMR检测谱图见附图2。由结果可知,CPP不含蛋白质,纯度高,氢谱检测发现CPP含有甲基和乙酰基,α/β异头氢信号位于4.5~5.5ppm,糖环质子信号位于3.3~4.4ppm,未发现有其他杂质信号峰。
表1.CPP的理化性质检测结果
实施例2
蛋白核小球藻多糖混合物CPP体外消化特征及益生元活性研究
1.材料和方法
(1)材料
蛋白核小球藻多糖混合物CPP,同实施例1所述方法制备。
3,5-二硝基水杨酸,国药集团化学试剂有限公司;胃蛋白酶、胃脂肪酶、胰酶、乙醇、蛋白胨、酵母提取物、氯化血红素、树脂天青、吐温80、维生素K1、L-半胱氨酸盐酸盐、胆汁盐、NaOH、NaCl、K2HPO4、KH2PO4、MgSO4、CaCl2、NaHCO3等所用试剂均为市售分析纯试剂。
(2)方法
模拟唾液消化:配制的模拟唾液包含氯化钠(NaCl,0.12g/L)、氯化钾(KCl,0.15g/L)、粘蛋白(1.0g/L)、α-淀粉酶(2.0g/L),用0.1M HCl调pH至7.0。取2mL CPP溶液(8mg/mL)与2mL模拟唾液混合,以唾液和未加唾液的CPP为对照。置于37℃水浴中消化,分别于0、0.5、1、2h取出1.2mL反应液,沸水浴10min灭酶活。3,5-二硝基水杨酸(DNS)法测定唾液消化后多糖中还原糖的含量;HPGPC法测定消化后多糖分子量的变化;HPLC测定游离单糖。
模拟胃液消化:分别称取NaCl 310mg,KCl 110mg,CaCl215 mg,NaHCO360 mg,100mL超纯水溶解,用1M HCl调pH至3配制胃电解质。于100mL胃电解质中加入胃蛋白酶23.6mg,胃脂肪酶25mg,混匀后以1M HCl调pH至3配制得到胃液。取9mL CPP溶液(8mg/mL)与9mL唾液混合,置于37℃水浴中消化,分别于0、2、4、6h取样1.2mL,沸水浴灭酶活,HPGPC法检测分子量变化;DNS法检测还原糖含量;PMP单糖衍生法检测单糖。
模拟胃肠液消化:分别称取NaCl 270mg,KCl 32.5mg,CaCl216.5 mg,50mL超纯水溶解配制肠电解质。取0.07g/mL胰酶溶液100mL与肠电解质100mL和胰蛋白酶13mg混匀,用0.2MNaOH调pH至7配制成肠液。上述样品胃液消化6h后,以0.1MNaHCO3调pH至7,以比例(10:3)加入肠液,充分混匀,37℃水浴反应,反复震荡,分别于0、2、4、6h取样1.2mL,沸水浴灭酶活,检测分子量变化、还原糖含量及游离单糖含量。
体外模拟肠道菌群消化:
基础培养基配制:将2g蛋白胨、4g酵母提取物、0.1g NaCl、0.04g K2HPO4、0.04gKH2PO4、0.01g MgSO4、0.01g CaCl2、2.0g NaHCO3、0.02g氯化血红素、0.46g L-半胱氨酸盐酸盐、0.5g胆汁盐、1.0mg树脂天青、2.0mL吐温80和10μL维生素K1溶于1L蒸馏水,混匀,用1MHCl将pH调至7,灭菌。
新鲜粪便样本由五名健康志愿者(两名女性,三名男性,年龄均在20~30岁)提供。志愿者没有胃肠疾病且至少3个月未接受过抗生素治疗。将5g新鲜粪便样品与45mL高压灭菌后的改性生理盐水溶液(含L-半胱氨酸盐酸盐0.5g/L,NaCl 9.0g/L)搅拌混合均匀,低速离心去除大的粪便颗粒,得到便悬浮液(10%),合并五位志愿者的粪便悬浮液待用。取1mL粪便悬浮液加入9mL 0.01g/mL的CPP或FOS基础培养基(pH 7.0)中,立即转入厌氧盒中,再加入厌氧产气包后置于温度为37℃的培养箱中模拟体外肠道微生物厌氧发酵。以FOS作为阳性对照,分别于6、12、24和48h取样分析检测pH、分子量、DNS、多糖含量、单糖组成、红外光谱及SCFA(每个时间点设三组平行)。
CPP发酵后的肠道微生物分析:发酵48h后,采用DNA试剂盒(Omega Biotek,GA,USA)按照说明书的方法提取不同组的DNA。NanoDrop 2000紫外可见分光光度计(ThermoScientific,Wilmington,USA)和1%琼脂糖凝胶电泳检测DNA。采用引物341F(5’-CCTACGGGNGGCWGCAG-3’)和806R(5’-GGACTACHVGGGTATCTAAT-3’)对微生物16S rRNAV3-V4高变区进行PCR扩增。对PCR产物进行纯化和定量,生成测序库。采用IlluminanaavoseqPE250平台对文库进行测序。
CPP发酵后的短链脂肪酸浓度分析:发酵上清液与等体积含2-乙基丁酸的0.2M盐酸溶液混合作为内标。采用Agilent DB FFAP色谱柱(30m×0.25mm×0.25μm),GC-MS分析混合物中SCFAs的组成和水平。
2.结果
CPP模拟消化结果见表2。在胃肠消化过程中,总糖和还原糖含量均无显著变化。此外,在胃肠消化过程中CPP的色谱图中未观察到游离单糖,表明胃液和肠液不能消化CPP,CPP具有益生元的基本特征。
体外模拟肠道菌群发酵的CPP色谱图及发酵液pH变化分别见附图3和4。CPP在发酵过程中其峰值比例逐渐下降,说明CPP可被粪便菌群缓慢利用。从表2可知,随着发酵时间的延长,发酵液中总糖和还原糖含量逐渐降低。发酵48h后,CPP残留量为44.20%,表明有一半的CPP被肠道微生物利用。CPP发酵速率低于传统益生元,可能与CPP的结构复杂性有关。
发酵后微生物的α-多样性分析结果见表3;发酵后微生物组成的OUT水平分析结果见附图5;发酵后微生物在门水平和属水平的相对丰度见附图6;发酵后微生物在操作分类单元(OUT)水平的LEfSe分析见附图7。α-多样性分析可知,发酵48h后,CPP保持了较好的肠道菌群多样性。CPP组的细菌群落丰富度高于FOS组。从门的水平看,CPP组的Bacteroidetes丰度增高,F/B比值(0.56)低于空白组(2.59)和FOS组。越来越多的研究表明,F/B比值的降低与肥胖风险的降低正相关。从属的水平看,FOS和CPP组的大肠杆菌-志贺氏菌、梭杆菌、克雷伯氏菌等有害菌的相对丰度显著降低。CPP处理后的优势属为Parabacteroides(40.96%),其次为Phascolarctobacterium(8.19%)和Bacteroides(7.57%),这些均为对人体健康有益菌群。OUT水平分析表明,CPP组中的优势菌群为Parabacteroides属(OTU000026和OTU000075)和2种Parabacteroides属(OTU000010和OTU000063),与上述结果一致。其中,狄氏副拟杆菌Parabacteroides distasonis可缓解多发性硬化症和肥胖症。此外,Phascolarctobacteriumfaecium(OUT000008)也是CPP组中的主要物种,能够促进丙酸的产生,对人类胃肠道有益;Faecalibacterium prausnitzii(OUT000056)在CPP组中也明显增加了,该菌具有显著的健康促进作用,特别是抗炎作用,被认为是健康肠道菌群的特征菌群之一。
不同发酵时间点的SCFAs水平见表4。由于CPP与FOS对肠道菌群的影响不同,部分SCFAs的水平变化趋势有明显差异。FOS组的乙酸和丙酸浓度在发酵前12h呈持续上升趋势,随后在其余时间逐渐下降。而CPP组的乙酸和丙酸浓度从0h持续升高至48h,最高浓度分别为18.968和9.617mM,高于FOS组。在FOS或CPP组中,总SCFAs的浓度变化趋势与他们的乙酸和丙酸的浓度趋势相似。CPP组发酵48h时总SCFAs含量增至36.076mM,分别是空白组和FOS组的2.0倍和1.4倍。这些结果表明,CPP能显著促进短链脂肪酸的产生,与传统益生元相比,其发酵和SCFAs的产生速度较慢,能更好地促进人体健康。
表2.CPP模拟体外消化或发酵的总糖及还原糖含量变化
表3.发酵后微生物的α-多样性分析结果
表4.不同发酵时间点的短链不饱和脂肪酸(SCFAs)水平
以上对本发明做了示例性的描述,应该说明的是,在不脱离本发明的核心的情况下,任何简单的变形、修改或者其他本领域技术人员能够不花费创造性劳动的等同替换均落入本发明的保护范围。
Claims (10)
1.一种蛋白核小球藻多糖混合物,其特征在于,按照下述步骤制备获得:
步骤一、取蛋白核小球藻干粉,热水浸提或加木瓜蛋白酶酶解处理获得提取液;
步骤二、加入淀粉酶水解淀粉类多糖,乙醇和/或丙酮沉淀得到多糖提取物;
步骤三、透析、超滤或凝胶过滤除小分子杂质;
步骤四、最后减压冷冻干燥获得目标产物。
2.根据权利要求1所述的一种蛋白核小球藻多糖混合物,其特征在于:所述的一种蛋白核小球藻多糖混合物由5种不同分子量的多糖构成,分子量范围为10,000~650,000Da。
3.根据权利要求2所述的一种蛋白核小球藻多糖混合物,其特征在于:所述的一种蛋白核小球藻多糖混合物的分子量范围为15,000~600,000Da。
4.根据权利要求1所述的一种蛋白核小球藻多糖混合物,其特征在于:所述的一种蛋白核小球藻多糖混合物的总糖含量以苯酚硫酸法检测为45%~75%,葡萄糖醛酸含量为10%~30%,不含蛋白质,易溶于水。
5.根据权利要求1所述的一种蛋白核小球藻多糖混合物,其特征在于:所述的一种蛋白核小球藻多糖混合物包含的单糖组成为甘露糖(Man)、核糖(Rib)、鼠李糖(Rha)、GlcA、Glc、半乳糖(Gal)、木糖(Xyl)和阿拉伯糖(Ara),其摩尔比为(1.2±0.5):(0.6±0.3):(1.6±0.5):(1.8±0.5):(1.1±0.5):(3.0±0.5):(0.4±0.3):(2.3±0.5)。
6.一种蛋白核小球藻多糖混合物的制备方法,其特征在于,按照下述步骤制备获得:
步骤一、取蛋白核小球藻干粉,热水浸提或加木瓜蛋白酶酶解处理获得提取液;
步骤二、加入淀粉酶水解淀粉类多糖,乙醇和/或丙酮沉淀得到多糖提取物;
步骤三、透析、超滤或凝胶过滤除小分子杂质;
步骤四、最后减压冷冻干燥获得目标产物。
7.根据权利要求6所述的一种蛋白核小球藻多糖混合物的制备方法,其特征在于:在步骤一中,取蛋白核小球藻干粉置于反应釜中加水提取,料液比为1:10~1:30,85~95℃提取1~3次,每次1~4h,离心并合并所得提取液。
8.根据权利要求6所述的一种蛋白核小球藻多糖混合物的制备方法,其特征在于:在步骤二中,所述淀粉酶终浓度为0.01%~0.5%,溶液pH为5.5~7.5,酶解温度为40~70℃,酶解时间为1~4h,淀粉KI试纸监测直至水解液不变蓝为止。
9.一种蛋白核小球藻多糖混合物作为新型益生元在调节肠道微生物、在口服制剂或食品添加剂或口服药物中的应用。
10.根据权利要求9所述的应用,其特征在于,所述一种蛋白核小球藻多糖混合物具有抵抗唾液、胃肠液的消化,能够被人肠道微生物酵解利用,提高人肠道微生物物种的丰度和多样性;增加有益菌水平,抑制有害菌增殖;能够促进短链脂肪酸(SCFAs)产生;发酵特征异于传统益生元为CPP发酵速率,SCFAs水平变化趋势异于传统益生元。
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